CN101248191A

CN101248191A - Method and kit for analyzing a target nucleic acid sequence

Info

Publication number: CN101248191A
Application number: CNA2006800309358A
Authority: CN
Inventors: R·T·巴纳德; G·R·巴尼特
Original assignee: CT FOR INNOVATIONS Pty Ltd
Current assignee: CT FOR INNOVATIONS Pty Ltd
Priority date: 2005-07-06
Filing date: 2006-07-06
Publication date: 2008-08-20
Also published as: AU2006265698A1; EP1910558A4; WO2007003017A1; JP2008544756A; CA2614042A1; KR20080047355A; US20080199872A1; EP1910558A1

Abstract

This invention discloses methods for determining the presence of a target nucleic acid sequence using oligonucleotides that cooperate in a nucleic acid processing reaction to produce a detectable signal. In some embodiments, the methods also facilitate quantification of a target nucleic acid sequence. The present invention further discloses kits that can be used to conduct the methods of the present invention.

Description

Analyze the method and the test kit of target nucleic acid sequence

Invention field

Present invention relates in general to analyze the method for target nucleic acid sequence.More particularly, the present invention relates to measure the analytical procedure that whether has target nucleic acid sequence, thereby described oligonucleotide cooperates the generation detectable signal with the nucleic acid processing reaction with oligonucleotide.In some embodiments, these methods help the quantitative assay target nucleic acid sequence.The invention still further relates to the test kit that can be used for implementing the inventive method.

Background of invention

Available energy detects the systems analysis nucleic acid molecule with other short making nucleic acid molecular hybridization, and described short nucleic acid molecule typically refers to probe, primer or oligonucleotide.Because can sequence and some subsequences by nucleic acid molecule distinguish nucleic acid molecule, also because nucleic acid molecule can with combine such as complementary sequence-specifics such as oligonucleotide, this is possible.

Polymerase chain reaction (PCR) is one of sensitive foranalysis of nucleic acids system.Hybridization between oligonucleotide and the complementary target nucleic acid sequence has formed archaeal dna polymerase and has started the polymeric site, and archaeal dna polymerase is from this site copy target nucleic acid sequence template and then generation and the new DNA chain of this target nucleic acid complementary then.Come DNA amplification by new chain of continuous copy and raw chains.The PCR primer tasteless nucleotide is mixed among the product D NA of amplification, identify the product D NA of amplification then.By the gel electrophoresis separation and with behind the chemical substance dyeing PCR product, can carry out visual observations to it usually.Perhaps, during without gel electrophoresis, can combine quantitatively or half-quantitative detection PCR product with the preferential of double-stranded PCR product by dyestuff.

Also there are other several method for nucleic acid analysis, comprise that ligase chain reaction (LCR), oligonucleotide connect the amplification of test, join dependency PCR (ligation dependent PCR), strand displacement amplification, branched DNA amplification, rolling circle amplification, transcriptive intermediate, increase based on the amplification and the hybridization signal of nucleotide sequence.

Although many method for nucleic acid analysis are arranged, yet the method for having developed seldom can be distinguished two or more nucleic acid targets of two or more nucleic acid targets or quantitative assay simultaneously.It is unreliable and insensitive usually to be designed for the method for analyzing many different nucleic acid targets simultaneously.Therefore, the few routine of method for nucleic acid analysis that can analyze multiple nucleic acid target (promptly multiple) simultaneously is used for current medical science, veterinary science or agricultural diagnosis (Yang etc., 2004, Lancet.InfectiousDiseases 4:337-48; Broude etc., 2001, Proc.Natl.Acad.Sci.U S is A.98:206-211; Chamberlain etc., 1988, Nucleic Acids Res.16:11141-11156; Edwards etc., 1994, PCRMethods Appl.3:S65-S75; Hacia etc., 1998, Genome Res.8:1245-1258; Li etc., 1996, Nucleic Acids Res.24:538-539; Stuven etc., 1996, Pharmacogenetics 6:417-421; VanOrsouw etc., 1998, Genomics52:27-36).

Therefore, extremely need exploitation can not only analyze a kind of target nucleic acid sequence, can also analyze the sensitive method of multiple target nucleic acid sequence (being multiple analysis) simultaneously.

Summary of the invention

Therefore, on the one hand, the invention provides the sequence method of target nucleic acid in the analytical sample.These methods generally include:

-in a reaction vessel, merge:

(1) not with the seizure oligonucleotide of target nucleic acid sequence hybridization (for example, fixed or be free in the solution);

(2) provide detectable signal and with the signal oligonucleotide (signalingoligonucleotide) of catching oligonucleotide hybridization;

(3) at least a mosaic type oligonucleotide, it comprises:

(a) with the first target sequence of the subsequence of target nucleic acid sequence hybridization; With

(b) with the caught sequence that is selected from following sequence hybridization:

(i) catch oligonucleotide; Or

(ii) signal oligonucleotide

(4) comprise at least a interoperability oligonucleotide of the second target sequence, the described second target sequence be selected from following sequence hybridization:

(a) subsequence different in the target nucleic acid sequence with the subsequence of the described first target sequence hybridization; Or

(b) subsequence of the complementary strand of target nucleic acid sequence, or

(c) at least a mosaic type oligonucleotide; With

(5) comprise the sample of nucleic acid;

-make the inclusion in this reaction vessel carry out the nucleic acid processing reaction to form reaction product (if having target nucleic acid sequence in the sample), wherein the reaction product that so forms comprises first chain and second chain, first chain comprises at least a mosaic type oligonucleotide or its extension products, second chain comprises at least a interoperability oligonucleotide or its extension products, thereby the caught sequence of having blocked the mosaic type oligonucleotide with catch oligonucleotide hybridization, and then make the signal oligonucleotide with catch oligonucleotide hybridization; With

The detectable signal of-detection signal oligonucleotide, the existence or the content of target nucleic acid in this signal indicating sample.

In some embodiments, the nucleic acid processing reaction is a polymerization dependency nucleic acid processing reaction, and its illustrative example comprises:

A) make the target sequence of mosaic type oligonucleotide and target nucleic acid sequence hybridization form first crossbred, wherein this target nucleic acid sequence extends to beyond the 3 ' terminal nucleotide of this mosaic type oligonucleotide with 3 '-5 ' direction, thereby determines the non-hybridization portion of this target nucleic acid sequence;

B) in the presence of polymerization agent and nucleotide precursor, use the non-hybridization portion of this target nucleic acid sequence to extend the mosaic type oligonucleotide of this first crossbred, thereby formed first duplex (duplex) that comprises first extension products and target nucleic acid sequence as template;

C) make this first duplex sex change to discharge target nucleic acid sequence from first extension products;

D) make the hybridization of the interoperability oligonucleotide and first extension products, thereby form second crossbred, wherein this first extension products extends to beyond the 3 ' terminal nucleotide of this interoperability oligonucleotide with 3 '-5 ' direction, thereby has determined the non-hybridization portion of this first extension products; With

E) in the presence of polymerization agent and nucleotide precursor, use this first extension products to extend the interoperability oligonucleotide of this second crossbred as template, thus formed comprise this first extension products and with the reaction product of this first extension products complementary, second extension products.

Step a)-e) should repeat one or many, between about 100 times of about 1-, generally between about 50 times of about 10-, more common between about 40 times of about 20-usually.In some embodiments, polymerization agent is primer dependent dna-polymerases (optional heat-staple), and its illustrative example comprises (Pfu) archaeal dna polymerase of fierce fireball bacterium (Pyrococcus furiosis), fireball bacterial classification (Pyrococcu sp.) GB-D (Psp) archaeal dna polymerase, Wo Shi fireball bacterium (Pyrococcus woesei) is archaeal dna polymerase (Pwo), thermus aquaticus (Thermus aquaticus) is archaeal dna polymerase (Taq), Bu Shi (Tbr) archaeal dna polymerase of hot bacterium (Thermus brocianus) of dwelling, Huang (Tfl) archaeal dna polymerase of hot bacterium (Thermusflavus) of dwelling, venting hole is had a liking for high temperature bacterium (Thermococcus litoralis) (Tli or Vent) archaeal dna polymerase, Thermotoga maritima (Thermotoga maritima) is (Tth) archaeal dna polymerase and their derivative of archaeal dna polymerase and thermus thermophilus (Thermus thermophilus) (Tma).

In some embodiments, polymerization agent in the step b) is a primer dependency reversed transcriptive enzyme, such as but not limited to: avian myeloblastosis virus (AMV), Moloney murine leukemia virus (MMLV) and thermus thermophilus (Thermus thermophilus) be archaeal dna polymerase and their derivative (Tth).

In some embodiments, the polymerization agent in the step e) is an archaeal dna polymerase, and it should be heat-staple.

In other embodiments, polymerization dependency nucleic acid processing reaction comprises:

I) but make the first target sequence of the first interoperability oligonucleotide of cyclisation and first subsequence hybridization of target nucleic acid sequence form first crossbred, wherein 5 ' the Nucleotide complementation of 3 ' Nucleotide of this first interoperability oligonucleotide and this first subsequence;

Ii) make the second target sequence of this first interoperability oligonucleotide and second subsequence hybridization of this target nucleic acid sequence form second crossbred, wherein this second subsequence is adjoined (adjacent to) this first subsequence;

Iii) the 5 ' Nucleotide of this first subsequence be connected reagent in the presence of, the first and second target sequences that connect this first interoperability oligonucleotide, first duplex that forms, this first duplex comprise the connection product of the first and second target sequences that comprise this first interoperability oligonucleotide of first and second subsequences of this target nucleic acid sequence and cyclisation form;

Iv) make this first duplex sex change connect product to discharge from target nucleic acid;

V) make the mosaic type oligonucleotide and be connected product hybridization to form the triple-crossing body;

Vi) in the presence of polymerization agent and nucleotide precursor, use the connection product to extend the mosaic type oligonucleotide of this triple-crossing body, thereby form first extension products as template;

Vii) make the second interoperability oligonucleotide and this first extension products hybridization with form the 4th crossbred and

Viii) (for example at polymerization agent, the primer dependent dna-polymerases, such as but not limited to φ 29DNA polysaccharase) exist down with nucleotide precursor, use this first extension products to extend the second interoperability oligonucleotide of the 4th crossbred as template, thus form comprise first extension products and with the reaction product of this first extension products complementary, second extension products.

In other embodiments, the nucleic acid processing reaction is a ligase enzyme dependency nucleic acid processing reaction, and its illustrative example comprises:

1. make first target sequence of mosaic type oligonucleotide and first subsequence of target nucleic acid sequence hybridize to form first crossbred wherein 5 ' the Nucleotide complementation of 3 ' Nucleotide of this mosaic type oligonucleotide and this first subsequence;

2. make the second target sequence of the first interoperability oligonucleotide and second subsequence hybridization of this target nucleic acid sequence form second crossbred, wherein this second subsequence is adjoined this first subsequence;

The 5 ' Nucleotide of this first subsequence be connected reagent in the presence of, connect this mosaic type oligonucleotide and this first interoperability oligonucleotide, first duplex that forms, it comprises first and second subsequences of this target nucleic acid sequence and comprises this mosaic type oligonucleotide simultaneously and the product that is connected of this first interoperability oligonucleotide;

4. make this first duplex sex change connect product to discharge from target nucleic acid; With

5. the mosaic type oligonucleotide and the first interoperability sequence that make the second interoperability oligonucleotide be connected product with this are partly hybridized to form reaction product.

In other illustrative example, ligase enzyme dependency nucleic acid processing reaction comprises:

First target sequence of the first mosaic type oligonucleotide and first subsequence of target nucleic acid sequence are hybridized to form first crossbred, wherein 5 ' the Nucleotide complementation of 3 ' oligonucleotide of this first mosaic type oligonucleotide and this first subsequence, and the 5 ' Nucleotide that can catch sequence can not connect;

B. make second subsequence hybridization of adjoining this first subsequence in the second target sequence of the second mosaic type oligonucleotide and the target nucleic acid sequence to form second crossbred, the 5 ' Nucleotide that wherein can catch sequence can not connect;

C. the 5 ' Nucleotide of this first subsequence be connected reagent in the presence of, connect this first mosaic type oligonucleotide and this second mosaic type oligonucleotide to form first duplex, this first duplex comprises first and second subsequences of this target nucleic acid sequence and comprises this first mosaic type oligonucleotide simultaneously and the product that is connected of this second mosaic type oligonucleotide;

D. make this first duplex sex change connect product to discharge from target nucleic acid; With

E. make the interoperability oligonucleotide be connected the first and second mosaic type oligonucleotide hybridizations of product with this to form reaction product.

In also having other embodiment, ligase enzyme dependency nucleic acid processing reaction comprises:

I. make the first target sequence of the first mosaic type oligonucleotide and first subsequence hybridization of target nucleic acid sequence form first crossbred, wherein the caught sequence that comprises of this first mosaic type oligonucleotide can with catch oligonucleotide hybridization, and 5 ' the Nucleotide complementation of 3 ' Nucleotide of this first mosaic type oligonucleotide and this first subsequence;

Ii. make second subsequence hybridization of adjoining this first subsequence in the second target sequence of the second mosaic type oligonucleotide and the target nucleic acid sequence form second crossbred, wherein the caught sequence that comprises of this first mosaic type oligonucleotide can with the signal oligonucleotide hybridization;

Iii. the 5 ' Nucleotide of this first subsequence be connected reagent in the presence of, connect this first mosaic type oligonucleotide and this second mosaic type oligonucleotide to form first duplex, this first duplex comprises first and second subsequences of this target nucleic acid sequence and comprises this first mosaic type oligonucleotide simultaneously and the reaction product of this second mosaic type oligonucleotide; With

Iv. make this first duplex sex change with release reaction product and target nucleic acid.

In some embodiments, step 1-5 or a-e or i-iv can repeat one or many, and be between about 100 times of about 1-, generally between about 50 times of about 10-, more common between about 40 times of about 20-usually.

Connect reagent and should be selected from T4DNA ligase enzyme, intestinal bacteria (Escherichia coli) dna ligase and the thread hot bacterium that dwells (Thermus filiformis) (Tfi) dna ligase and their derivative.

On the other hand, the invention provides the test kit that following material is housed:

(2) provide detectable signal and with the signal oligonucleotide of catching oligonucleotide hybridization;

(3) at least a mosaic type oligonucleotide, it comprises:

(i) catch oligonucleotide; Or

(ii) signal oligonucleotide

(b) subsequence of the complementary strand of target nucleic acid sequence, or

(c) at least a mosaic type oligonucleotide.

In some embodiments, test kit also is equipped with (5) one or more polymerizations and/or connects reagent.

In some embodiments, any one or more component shown in (1)-(5) (for example, 1,2,3,4 or 5) is a lyophilized form.(1)-(5) any two or various ingredients (for example, 2,3,4 or 5) shown in should be the forms of mixture.Perhaps they can be in independently in the container.

In some embodiments, catch oligonucleotide and be fixed on (for example, the surface of particulate or pearl, nano thread (nanowire), diagnosis band (diagnostic strip) or reaction vessel) on the solid surface.

In some embodiments, two or more are caught oligonucleotide and be fixed into the form of catching oligonucleotide arrays.

The accompanying drawing summary

Fig. 1: the sepharose photo that shows the product that is generated from following Australian influenza A (Australian Influenza A) isolate PB2 constant gene segment C by RT-PCR: H5N3 (+), H5N3, H11N6, H7N7, H12N9, H7N7, H4N4, H6N5 and H9N2.

Fig. 2: the synoptic diagram of a kind of embodiment of the inventive method of employing polymerase chain reaction (PCR).(Fig. 2 A) fixed is caught oligonucleotide (B); (Fig. 2 B) comprises the signal oligonucleotide (B ") of signal reagent (C); (Fig. 2 C) comprises the mosaic type oligonucleotide that can catch sequence (B ') and target sequence (E ') and interoperability oligonucleotide (F '); (Fig. 2 D) target nucleic acid sequence (E); (Fig. 2 E) comprised the mosaic type oligonucleotide of first extension products (F) and the crossbred of target nucleic acid sequence; (Fig. 2 F) mosaic type oligonucleotide and target nucleic acid sequence that comprises extension products and the crossbred that comprises the interoperability oligonucleotide of second extension products (G); (Fig. 2 G) fixed is caught oligonucleotide and the crossbred (that is positive signal) that comprises the signal oligonucleotide of signal reagent; (Fig. 2 H) fixed is caught the crossbred (that is negative signal) of oligonucleotide and mosaic type oligonucleotide.

Fig. 3: the synoptic diagram of a kind of embodiment of the inventive method of employing rolling circle amplification (RCA).

Fig. 4: the synoptic diagram that adopts a kind of embodiment of the inventive method that connects chain reaction (LCR).

Fig. 5: the synoptic diagram that adopts a kind of embodiment of the inventive method that connects chain reaction (LCR).

Fig. 6: the synoptic diagram that adopts a kind of embodiment of the inventive method that connects chain reaction (LCR).

Fig. 7: show the diagram that adopts end point determination (end point detection) to analyze H7N7 influenza A cDNA.Utilize the 0.5pmolPCR-label primer (PCR-TAG primer) of 50 μ L carry out 35 take turns PCR after, detect the 450nm absorbancy

Fig. 8: show titration experiments result's diagram, the optimum amount of PCR-label primer in the embodiment of this experiment employing end point determination step measurements the inventive method.35 take turns the sample absorbancy that detects 450nm behind the pcr amplification.Darker point shows the absorbancy when the PCR target exists, brighter some display background absorbancy.

Fig. 9: show the agarose electrophoresis of PCR product and the photo of ethidium bromide staining, described PCR product increases according to identical test shown in Figure 8 and measures the optimum amount of PCR-label primer.In various situations, the consumption of reverse primer is 4 times of PCR-label primer.Negative control does not contain starting template, and PCR reaction cumulative volume is 50 μ l.The swimming Taoist monastic name is appointed as: M, Hyper Ladder II (supramolecule amount gradient II); (1) the PCR product of use 1 picomole PCR-label primer and H7N7 template; (2) the PCR product of use 0.5 picomole PCR-label primer and H7N7 template; (3) the PCR product of use 0.25 picomole PCR-label primer and H7N7 template; (4) the PCR product of use 1 picomole PCR-label primer negative control; (5) the PCR product of use 0.5 picomole PCR-label primer negative control; (6) the PCR product of use 0.25 picomole PCR-label primer negative control.

The sequence summary

SEQ ID NO	Sequence
SEQ ID NO	Sequence	12 34567 8910	5′-CTT TAA TCT CAA TCA ATA CAA ATC-3′5′-CTT TAA TCT CAA TCA ATA CAA ATC AG(C/T)TCI TC(C/T) TT(C/T)AG(C/T)TT(C/T)GG-3′5′-AGT AT(T/C) CTC AT(T/C) CC(T/A)GAN CC-3′5′-GAT TTG TAT TGA TTG AGA TTA AAG-3′5′-CTT TAA TCT CAA TCA ATA CAA ATC TTG CAG CIG CIC CAC CIG-3′5′-AIC AIA GIA GIA TGC AGT-3′5′-AIC AIA GIA GIA TGC AGT TCA TAA GAC TCG TCA TGT CTC AGC AGC TTCTAA CGG TCA CTA ATA CGA CTC ACT ATA GG TTG CAG CIG CIC CAC CIG-3′5′-CTT TAA TCT CAA TCA ATA CAA ATC GCT GAG ACA TGA CGA GTC-3′5′-AAT ACG ACT CAC TAT AGG-3′5′-CTT TAA TCT CAA TCA ATA CAA ATC GAI GTI AGI GAI ACI CAI GG-3′

Detailed Description Of The Invention

1. definition

Unless otherwise defined, all technology used herein have with those skilled in the art with scientific terminology and understand identical connotation. Although any method similar or of equal value available and described herein and material are implemented or check the present invention, preferred method as herein described and material. Be purpose of the present invention, following term is as giving a definition.

Use article " " and " one " to refer to the grammatical object of one or more (that is, at least one) these articles herein. For example, " element " element of expression or more than one element.

" pact " represents that certain quantity, level, numerical value, number, frequency, percentage, size, size, consumption, weight or length compares with reference quantity, level, numerical value, number, frequency, percentage, size, size, consumption, weight or length, and 30,25,20,25,10,9,8,7,6,5,4,3,2 or 1% difference is arranged at most.

Term " amplification " or " nucleic acid amplification " or " amplified reaction " refer to produce the biochemical reaction of many polynucleotide copies of certain concrete target nucleic acid sequence. If target nucleic acid sequence is strand, then this reaction can produce complementary series. In some embodiments, this reaction is PCR (PCR) or the similar reaction of using polymerase copy nucleotide sequence, for example amplification (TMA) of helicase dependent amplification (HAD), transcriptive intermediate, strand displacement amplification (SDA), the amplification (NASBA) based on nucleotide sequence, rolling circle amplification (RCA) and reverse transcriptase polymerase chain reaction (RT-PCR). It is to cause (startup) to react required that the target nucleic acid sequence of oligonucleotides and single stranded form is hybridized formed double-stranded region. In other embodiments, term " amplification " or " nucleic acid amplification " or " amplified reaction " refer to use ligase or similar enzyme to come the biochemical reaction of covalently bound two oligonucleotides or two oligonucleotides subsequences, for example ligase chain reaction (LCR). When two oligonucleotides or oligonucleotides subsequence in target nucleic acid sequence adjoin site hybridization the time, ligase can connect the two. Perhaps, if two oligonucleotides or oligonucleotides subsequence are separating the site hybridization of one or more nucleic acid residues, be that they do not adjoin, then with polymerase the strand zone between the double-stranded region is changed into double-stranded region, then connect the oligonucleotides adjoin to form continuous double-stranded region with ligase.

Term " can catch sequence " and refer to can with the nucleotide sequence of another nucleic acid array hybridizing. In some embodiments, can catch sequence and catch oligonucleotide hybridization, the latter is fixed in holder (for example, the surface of solids) or is free in the solution in exemplary example.

Term " seizure oligonucleotide arrays " expression is fixed on a plurality of seizure oligonucleotides of known discontinuous position on the surface of solids. Surface for reaction vessel or diagnosis band catches oligonucleotides and can be arranged in the two-dimensional space addressing array, for example 2 * 2 arrays. Perhaps, catch oligonucleotides and can be arranged in tubular configuration. In other embodiments, catch on the inner surface or outer surface that oligonucleotides is arranged in the bidimensional of any conventional topology structure or three-dimensional reaction vessel. Nucleic acid array is known in the art, can classify in many ways; Comprise oldered array (for example, differentiating the ability of the chemical substance in discontinuous site) and random array. Oldered array includes but not limited to the array that adopts following technology to make: (A Fumai bends this genetic chip of gram (Affymetrix GeneChip to photolithographic techniques^TM)), point sample technology (Xin Taini (Synteni) and other array), printing technology (Xiu Laite packard array (Hewlett Packard) and Marc rosset array (Rosetta)), three-dimensional " gel mat " array etc. Also can use the liquid array, i.e. cubical array method, for example flow cytometry. When adopting flow cytometry, catching oligonucleotides should be fixed in such as holders such as microballoons.

In some embodiments, oldered array is included in the array that known location contains nucleic acid. That is, as herein described catch sequence or catch oligonucleotides be fixed on known location on the substrate. Known or the original known site of " known " positional representation.

Term used herein " mosaic type oligonucleotides " refers to comprise the oligonucleotides of at least two nucleotide sequences or part, and the position of described at least two nucleotide sequences or part or connected mode are not natural generations.

Term " complementation " refers to the nucleotide sequence relevant by base pairing rules with " complementarity ". For example, sequence " A-G-T-C " and sequence " T-C-A-G " complementation. Complementation can be " part is complementary ", wherein only has some nucleic acid bases to mate according to base pairing rules. Therefore, to need not be completely to complementarity; Can have can affect any amount of base of hybridizing between First ray and the second sequence and not mate. Yet, also can not hybridize under the minimum hybridization conditions of stringency if sudden change quantity is too large, this sequence is not complementary series. Therefore, " basically complementary " expression First ray and the second sequence are enough complementary, thereby can hybridize under selected reaction condition. The well known relation that is enough to realize specific complementarity and hybridization stringency. Therefore, can use basically complementary sequence in any analytical method of the present invention. For example, this sequence can be maybe can containing 1 and not mating to a plurality of of complete complementary, as long as hybridization conditions is enough to distinguish target sequence and non-target sequence. Therefore, the homogeny percentage of complementary sequence can be 100,99,98,97,96,95,94,93,92,91,90,89,85,80,75 or lower basically. Perhaps, can have " fully " or " totally " complementarity between the nucleic acid. Complementary degree between the nucleic acid chains has a significant effect to efficient and the intensity of hybridizing between the nucleic acid chains.

Unless needs are arranged in addition, otherwise the word in this specification " comprises ", " containing " and " comprising " be interpreted as having hinted and comprise a described step or element or step group or element group, but also do not get rid of any other step or element or step group or element group.

The pairing of " hybridization " used herein expression complementary nucleotide sequence has produced DNA-DNA, DNA-RNA or DNA-PNA crossbred. Complementary base sequence is those sequences relevant by base pairing rules. For DNA, A and T pairing, C and G pairing. For RNA, U and A pairing, C and G pairing. Also can use base inosine (I). Inosine can form base pairing with C or A or G or T (stability is descending). In this, term used herein " coupling " and " not mating ", refer to the hybridization ability of pairing nucleotides in the complementary nucleic acid chain. The nucleotides of coupling can effectively be hybridized for example above-mentioned classical A-T and G-C base pairing. Not mating is other combination of the nucleotides that can not effectively hybridize.

" separation " expression does not contain under the nature material with its component that normally accompanies basically or in fact. For example, " oligonucleotides of separation " used herein refers under naturally occurring state the oligonucleotides that is purified into the sequence with its side joint, for example, removed the dna fragmentation of its normal contiguous nucleotide sequence.

Term used herein " oligonucleotides " refers to by the polymer by the continuous a plurality of nucleotide units of phosphodiester bond (or its relevant structural variant or synthetic analogues) (deoxyribonucleotide or ribonucleotide or its relevant structural variant or synthetic analogues) formation. Therefore, although term " oligonucleotides " is often referred to wherein nucleotides and the connecting key nucleotide polymer that is natural generation, but should be appreciated that in the scope of this term also to comprise various analogs, include but not limited to: peptide nucleic acid (PNA), phosphoramidate, thiophosphate, methyl-phosphonate, 2-O-methylribose nucleic acid etc. The definite size of this molecule can be because of the concrete difference of using. Oligonucleotides is usually shorter, general about 10-30 nucleotides, and this term also can refer to the molecule of any length, although term " polynucleotides " or " nucleic acid " are generally used for large oligonucleotides.

Term used herein " oligonucleotides ", " polynucleotides " or " nucleic acid " refer to DNA, cDNA, RNA, mRNA, cRNA or PNA. This term is often referred to length greater than the oligonucleotides of 30 nucleotides.

When " primer " expression and DNA or the pairing of RNA chain, can in the presence of suitable polymerization agent, start the synthetic oligonucleotides of primer extension product. For obtaining maximum amplification efficiency, primer is strand normally, but can also be double-stranded. The necessary long enough of primer, thus the synthetic of extension products can in the presence of polymerization agent, be caused. The length of primer depends on many factors, comprises application, the temperature that adopts, template reaction condition, other reagent and primer source. For example, according to the complexity of target sequence, Oligonucleolide primers contains 15-35 or more nucleotides usually, although it can only contain less nucleotides. Primer can be large polynucleotides, for example about 200 nucleotides-several kilobase or more. Can select primer, make itself and the sequence " basically complementary " that designs on the template of hybridizing with it, and can be used as the synthetic site of startup. The complementarity of " basically complementary " expression primer is enough to hybridize with target nucleotide sequences. Preferably do not contain mispairing between the template that primer and design are hybridized with it, but this is optional. For example, non-complementary nucleotides can be connected in 5 ' end of primer, remaining primer sequence and template are complementary. Perhaps, incomplementarity nucleotides or non-complementary nucleotides section can be inserted in the primer, prerequisite is that primer sequence is enough complementary with the template sequence that will hybridize, thereby is formed for the template of the extension products of synthetic primer.

The term that is used for sequence relation between the description two or more pieces nucleotide sequence comprises " reference sequence ", " comparison window ", " sequence homogeny ", " sequence homogeny percentage " and " substantially the same ". The length of " reference sequence " is 10 at least, but often is 15-20, often is at least 25 monomer unit (being nucleotides). Because two nucleotide sequence can respectively contain (1) similar sequence is (namely between two polynucleotides, be the part of whole nucleotide sequence), (2) different sequence between two nucleotide sequences, general passing through compared the sequence of nucleotide sequence with the regional area of evaluation and comparative sequences similitude in " comparison window ", thereby carries out sequence relatively between two (or many) nucleotide sequences. " comparison window " refers at least 50, and about 50-is about 100 usually, and the conceptual section of about 150 continuous positions of more common about 100-after wherein two sequences carries out the best comparison, is made comparisons a sequence reference sequence identical with continuous position quantity. For the best comparison two sequences, to compare with reference sequence (its do not comprise add or disappearance), comparison window can comprise about 20% or interpolation still less or disappearance (that is, room). For comparing certain comparison window, can pass through computer execution algorithm [(GAP, BESTFIT, FASTA and the TFASTA of Wisconsin science of heredity software kit 7.0 editions (Wisconsin Genetics Software Package Release 7.0), science of heredity calculates unit (Genetics Computer Group), 575 Science Drive Madisons, the state of Wisconsin, the U.S.)] compare (that is the percent homology that, obtains in comparison window is the highest) next best aligned sequences or by the best that observation and selected the whole bag of tricks produce. Also can be with reference to BLAST family program, such as Altschul etc., 1997, Nucl.Acids Res.25:3389 is disclosed. Sequence analysis discuss visible Ausubel etc. in detail, " " up-to-date molecular biology method " (Current Protocols in Molecular Biology) ", John Wei Li father and son company (John Wiley Sons Inc), 1994-1998,19.3 parts in the 15th chapter.

The container of term " reaction vessel " fingering row the inventive method. Reaction vessel can be any container that is applicable to the standard molecular biology reaction, therefore can be fit to the material construction of this application. This material can be natural or synthetic, but the perhaps natural and synthetic material formation of coupling. The exemplary materials that can be used for making up reaction vessel comprises plastics (for example, Merlon, polystyrene and polypropylene), glass etc. Particularly preferred reaction vessel of the present invention comprises conventional PCR test tube and microtiter plate, for example 96-hole microtiter plate. In some embodiments of the present invention, be fixed on the inner surface of reaction vessel catching oligonucleotides. In these embodiments, reaction vessel should make up with transparent material, thereby can be from reaction vessel external detection signal oligonucleotides and the hybridization that catches oligonucleotides. In concrete embodiment, reaction vessel is characterised in that it has basically flat surface. Perhaps, reaction vessel is characterised in that it has the basically surface of tubulose, is about to the two-dimensional plane thin plate and is rolled into three-dimensional tube shape configuration. The available this configuration more seizure oligonucleotides of high surface area for example is fixed in " circulation " cell. In other embodiments, reaction vessel is microfluidic devices.

In this article, term " sequence homogeny " and " homogeny " are used interchangeably, and it refers to that nucleotide sequence is according to nucleotides-nucleotides or amino acid-amino acid whose same degree on comparison window. Therefore, " sequence homogeny percentage " calculates by following steps: the sequences that compare two best comparisons in comparison window, (for example measure two sequences amplifying nucleic acid base, A, T, C, G, I) identical positional number to be to obtain the number of matched position, with the number of matched position divided by the total number of positions in the comparison window (namely, window is big or small), thereby being multiply by 100, the result obtains sequence homogeny percentages. For purpose of the present invention, " sequence homogeny " is interpreted as representing the used standard default value of reference manual of enclosing with software, by DNASIS computer program (2.5 versions that are used for windows; Available from soft project Co., Ltd of Hitachi (Hitachi Software engineering Co., Ltd.), southern San Francisco, California, the U.S.) " match-percentage " calculated.

Temperature and ionic strength conditions during " stringency " used herein refers to hybridize, and whether have some organic solvent. Stringency is higher, and the complementary degree between the nucleotide sequence of hybridizing is higher.

" stringent condition " used herein refers to following temperature and ionic strength: only have basically complementation or complementary base ratio high under this condition, preferably have the polynucleotides of accurate complementarity and oligonucleotides and just can hybridize and can produce amplified production in some embodiments. Desired stringency depends on nucleotide sequence, and depends on the various components that exist during the hybridization, and great changes have taken place with used nucleotide analog usually for stringency. Those skilled in the art know stringent condition. For the probe that oligonucleotides is used as in the hybridization reaction, stringent condition is selected usually than hanging down about 10-20 ℃ in the calculating pyrolysis chain temperature (Tm) of determining concrete sequence under ionic strength and the pH. Tm is the temperature (under definite ionic strength and pH) of 50% target sequence and complementary probe hybridization. Those skilled in the art know the calculating of Tm. Utilize following formula best computational length be shorter than the Tm:Tm=4 (wG+xC)+2 (yA+zT) of the oligonucleotides of 14 base residues-16.6*log10 (0.050)+16.6*log10 ([Na+]), wherein w, x, y, z are respectively the numbers of bases G in the sequence, C, A, T, and [Na+] is salinity. For the oligonucleotides of being longer than 13 base residues, by Breslauer etc. (1986, Proc.Nat.Acad.Sci. 83:3746-50) described arest neighbors formula (nearest neighbour formulae), but the numerical computations Tm that (1996, Nucl.Acids Res.24:4501-4505) such as use Sugimoto announces. The numerical value of RNA thermodynamic behaviour can be taken from (1998, Biochemistry 37:14719-14735) such as Xia. Should be appreciated that oligonucleotide probe or primer at least can be under low stringency conditions, preferably at least under medium stringent condition, more preferably under high stringent condition, hybridize with target sequence. The low stringency condition of Probe Hybridization reaction described herein comprises and comprises: 42 ℃ of hybridization conditions are at least about 1%v/v-at least about the formamide of 15%v/v with at least about the salt of 1M-at least about 2M; 42 ℃ of wash conditions are at least about 1M-at least about the salt of 2M. Low stringency condition also comprises: 65 ℃ of hybridization conditions are 1% seralbumin (BSA), 1mM EDTA, 0.5M NaHPO4 (pH7.2), 7%SDS; Wash conditions is (i) 2 * SSC under the room temperature, 0.1%SDS; Or (ii) 0.5%BSA, 1mM EDTA, 40mM NaHPO4 (pH7.2), 5%SDS. The medium stringent condition of Probe Hybridization reaction comprises and comprises: 42 ℃ of hybridization conditions are at least about 16%v/v-at least about the formamide of 30%v/v with at least about the salt of 0.5M-at least about 0.9M; 42 ℃ of wash conditions are at least about 0.5 M-at least about the salt of 0.9M. Medium stringent condition also comprises: 65 ℃ of hybridization conditions are 1% bovine serum albumin(BSA) (BSA), 1mM EDTA, 0.5M NaHPO4 (pH7.2), 7%SDS; 42 ℃ of wash conditions are (i) 2 * SSC, 0.1%SDS; Or (ii) 0.5%BSA, 1mM EDTA, 40mM NaHPO4 (pH7.2), 5%SDS. The high stringent condition of Probe Hybridization reaction comprises and comprises: 42 ℃ of hybridization conditions are at least about 31%v/v-at least about the formamide of 50%v/v with at least about the salt of 0.01M-at least about 0.15M; 42 ℃ of wash conditions are at least about 0.01M-at least about the salt of 0.15M. High stringent condition also comprises: 65 ℃ of hybridization conditions are 1%BSA, 1mM EDTA, 0.5M NaHPO4 (pH7.2), 7%SDS; Wash conditions more than 65 ℃ is (i) 0.2 * SSC, 0.1%SDS; Or (ii) 0.5%BSA, 1mM EDTA, 40mM NaHPO4 (pH7.2), 1%SDS. Other stringent condition of well known Probe Hybridization reaction. The known various factors of can regulating of technical staff is optimized the hybridization specificity. Can optimize the stringency of final washing to guarantee high hybridization degree. Detailed example is referring to " up-to-date molecular biology method " (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (the same), 2.10.1-2.10.16 page or leaf and " molecular cloning: laboratory manual " (MOLECULAR CLONING.A LABORATORY MANUAL) volumes such as () Sambrook (cold spring port publishing house, 1989), 1.101-1.104 chapter.

Phrase " target nucleic acid sequence " refers to interested any nucleotide sequence. It can be complete gene or its part. Therefore, target nucleic acid sequence can be to comprise genetic mutation, such as but not limited to certain gene part of nucleotides insertion, disappearance and SNP (SNP). Target nucleic acid sequence can also be complete gene or the nucleic acid of its part coding. Therefore, the target nucleic acid sequence considered of the present invention comprises DNA, cDNA, RNA, mRNA and cRNA. Target nucleic acid sequence can also be natural generation or synthetic nucleic acid molecules.

2. analyze the method for target nucleic acid sequence

The invention provides the method for analyzing target nucleic acid sequence, for example measure it and whether exist or quantity. The method is utilized multiple oligonucleotides and the cooperation of nucleic acid processing reaction, thereby produces detectable signal in the presence of target nucleic acid sequence. Described multiple oligonucleotides comprises:

(1) optional be fixed on the surface of solids and not with the seizure oligonucleotides of target nucleic acid sequence hybridization;

(2) provide detectable signal and with the signal oligonucleotides that catches oligonucleotide hybridization;

(3) can be used as in some embodiments at least a mosaic type oligonucleotides of primer, it comprises:

(i) catch oligonucleotides; Or

(ii) signal oligonucleotides; With

(4) comprise at least a collaborative oligonucleotides of the second target sequence, it can be used as primer in some embodiments, described the second target sequence be selected from following sequence hybridization:

(a) subsequence different from the subsequence of described the first target sequence hybridization in the target nucleic acid sequence; Or

(b) subsequence of the complementary strand of target nucleic acid sequence, or

(c) at least a mosaic type oligonucleotides.

The following product (if having target nucleic acid sequence in the sample) that nucleic acid processing reaction of the present invention obtains: it comprises the first chain and the second chain, the first chain comprises at least a mosaic type oligonucleotides or its extension products, the second chain comprises at least a collaborative oligonucleotides or its extension products, thereby the caught sequence of having blocked chimeric oligonucleotide with catch oligonucleotide hybridization, and then so that the signal oligonucleotides with catch oligonucleotide hybridization and provide whether target exists or the signal of quantity. When target nucleic acid sequence did not exist, the nucleic acid processing reaction can not occur, thus so that the caught sequence of mosaic type oligonucleotides and seizure oligonucleotide hybridization and disabling signal.

3. nucleic acid processing reaction

The inventive method can adopt any nucleic acid processing method that can produce above-mentioned product. Exemplary nucleic acid processing method comprises nucleic acid amplification. The exemplary process of well known nucleic acid amplification includes but not limited to: PCR (referring to, such as Saiki etc., 1985, Science, 230:1350-1354; Mullis etc., 1987, Methods Enzymol 155:335-350), strand displacement amplification (SDA and multiple SDA (MSDA); Referring to, for example United States Patent (USP) 5,422,252 and Little etc.), rolling circle amplification (RCA; Referring to, such as Liu etc., 1996, J.Am.Chem.Soc 118:1587-1594 and United States Patent (USP) 5,854,033 and United States Patent (USP) 6,642,034), based on the amplification (NASBA of nucleotide sequence; Referring to, such as Sooknanan etc., 1994, Biotechniques 17:1077-1080), ligase chain reaction (LCR; Referring to, for example WO 89/09835) and Q β replicase amplification (referring to, such as Tyagi etc., 1996, the same). Also can utilize the various alternatives of these technology, Syvanen Anne-Christine for example, sum up (2001, the same). In the method, can utilize any available feature of different amplified reactions to make up to improve sensitivity and/or the specificity of the method.

In some embodiments, nucleic acid processing reaction of the present invention is based on polymerase dependence nucleic acid amplification reaction. This type of exemplary amplification is PCR (PCR), wherein when an oligonucleotides oligonucleotides complementary with it in a pair of oligonucleotides separates, can be used as the extension products that template is synthesized this another oligonucleotides of centering by its synthetic extension products. Yet, one skilled in the art will appreciate that and usually utilize heat-staple primer dependence polymerase that used concrete PCR method is depended in the selection of polymerase usually.

As shown in Figure 2, in this type of exemplary example, form the first crossbred between the target sequence of mosaic type oligonucleotides and the target nucleic acid sequence. Then extend the target sequence with polymerization agent, this reagent can be primer dependent dna-polymerases or primer dependence reverse transcriptase. The effect of this kind of enzyme is with ribonucleoside triphosphote (for example, deoxyribonucleotide triphosphoric acid; DNTP) mix in the target sequence extension of crossbred, this effect of mixing can the crossbred of coupling pairing has selectively to containing between 5 ' terminal nucleotide of 3 ' terminal nucleotide of Oligonucleolide primers and target nucleotide sequences fully, perhaps non-selectivity, no matter this pairing coupling whether fully. In this, know some enzymes, for example eucaryon primer dependent dna-polymerases and birds myeloma virus (AMV) reverse transcriptase does not have the active (exonuclease of 3 ' error correction^-(exo ^-)), therefore only extend the nucleotides that contains between 5 ' terminal nucleotide of 3 ' terminal nucleotide of oligonucleotides and target nucleotide sequences combination in the complete crossbred of coupling. Perhaps, other binding reagents, for example the primer dependent dna-polymerases in protokaryon source comprises that for example the Klenow fragment of e. coli dna polymerase I has error correction activity (exonuclease⁺(exo ⁺)), therefore do not show that it is selective, although can buy from goods providers the modification of this kind of enzyme that lacks this error correction activity. Yet oligonucleotides extends use 3 ' exo^-Polymerization agent is normally desirable. Those skilled in the art know the suitable polymeric reagent that the nucleic acid processing reaction of PCR-based can be used. For the round that repeats PCR need not additionally to add archaeal dna polymerase, archaeal dna polymerase should be heat-staple, includes but not limited to: fierce fireball bacterium (Pyrococcus furiosis) is archaeal dna polymerase (Pfu), fireball bacterial classification (Pyrococcus sp.) GB-D (Psp) archaeal dna polymerase, Wo Shi fireball bacterium (Pyrococcus woesei) is archaeal dna polymerase (Pwo), thermus aquaticus (Thermus aquaticus) is the DNA polymerase (Taq), Bu Shi (Tbr) archaeal dna polymerase of hot bacterium (Thermus brocianus) of dwelling, Huang (Tfl) archaeal dna polymerase of hot bacterium (Thermus flavus) of dwelling, steam vent is had a liking for high temperature bacterium (Thermococcus litoralis) (Tli or Vent) DNA polymerase, Thermotoga maritima (Thermotoga maritima) is (Tth) archaeal dna polymerase and their derivative of archaeal dna polymerase and thermus thermophilus (Thermus thermophilus) (Tma). The suitable reverse transcriptase that the present invention is used, such as but not limited to: AMV (AMV), MMLV (MMLV) and thermus thermophilus (Thermus thermophilus) be archaeal dna polymerase and their derivative (Tth). The other factors of selective polymerization reagent comprises that the nucleic acid in the sample is DNA or RNA (that is, generally only having reverse transcriptase deoxynucleoside triphosphate effectively can be mixed in the extension products of RNA template).

The target sequence of utilizing target nucleic acid sequence to extend the mosaic type oligonucleotides as template can produce the duplex that contains the first extension products and target nucleic acid sequence. Then by sex change this first extension products is separated with target nucleic acid sequence, thereby so that the collaborative oligonucleotides can be hybridized with this first extension products, therefore formed the second crossbred. Then, extend this collaborative oligonucleotides with this first extension products as template as mentioned above. Because this first extension products comprises this mosaic type oligonucleotides, the second duplex that forms in this comprises this first extension products and complementary with this first extension products in stage, thus also with the second extension products of this mosaic type oligonucleotides complementation. This second duplex is corresponding to product of the present invention. With the sequence that can catch the sequence complementation be " blocking-up " sequence, its caught sequence that is used for blocking-up mosaic type oligonucleotides with catch oligonucleotide hybridization, thereby so that the signal oligonucleotides with catch oligonucleotide hybridization.

In other embodiments, the polymerase dependent amplification comprises rolling circle amplification (RCA), wherein Oligonucleolide primers can be connected with the hybridization of circular nucleic acid molecule, be cyclisation, archaeal dna polymerase (usually having the strand displacement activity) utilizes this circular nucleic acid molecule as synthetic the first extension products of template. Extension products is the longer nucleic acid molecule normally, and it contains a plurality of and repetitive sequence template circular nucleic acid complementary element. For PCR, in the presence of the complementary oligonucleotide primer, the first extension products can be used as subsequently template and synthesizes other extension products, thereby can increase primary template circular nucleic acid molecule.

As shown in Figure 3, in this type of exemplary example, form the first crossbred between collaborative oligonucleotides and the target nucleic acid sequence, thus can be after hybridization this collaborative oligonucleotides of cyclisation. The collaborative oligonucleotides has attachable end (E as shown in Figure 3₁' and E₂'), thereby available ligase catalysis connects 5 ' and 3 ' terminal (being specified in hereinafter) of this collaborative oligonucleotides. Contain the mosaic type oligonucleotides that can catch sequence (B ') and the collaborative extension products hybridization of cyclisation, cause the generation with enzymatic the first extension products of strand displacement polymerization. Another collaborative oligonucleotides of adding and the first extension products regional complementarity (F '), if cycling probe obtains connecting, then can cause the second extension products and produce. The caught Sequence complementation of the second extension products and mosaic type oligonucleotides. " blocking-up " sequence with the sequence that can catch the sequence complementation in the second extension products, the caught sequence of its blocking-up mosaic type oligonucleotides with catch oligonucleotide hybridization, thereby so that signal oligonucleotides and seizure oligonucleotide hybridization.

The archaeal dna polymerase that is suitable for the RCA that the present invention considers should be able to be replaced the chain with the template strand complementation, and this is called strand displacement, and these archaeal dna polymerases lack 5 ' to 3 ' exonuclease activities. Synthetic many parts of copies of collaborative oligonucleotides that connect need strand displacement. If have 5 ' to 3 ' exonuclease activity, then can destroy synthetic chain. It also is desirable that the used archaeal dna polymerase of the inventive method has the height continuation. The ability of carrying out rolling-circle replication by assessment the inventive method used archaeal dna polymerase is not difficult to measure it and whether is applicable to the inventive method. The exemplary circular DNA polymerase that rolls comprises bacteriophage Φ 29 archaeal dna polymerases (United States Patent (USP) 5,198,543 and 5,001,050), bacteriophage M2 archaeal dna polymerase (Matsumoto etc., Gene 84:247 (1989)), bacteriophage Φ PRD1 archaeal dna polymerase (Jung etc., Proc.Natl.Acad.Sci.USA 84:8287 (1987)), VENT.RTM.DNA polymerase (Kong etc., J.Biol.Chem.268:1965-1975 (1993)), Klenow fragment (the Jacobsen etc. of DNA polymerase I, Eur.J.Biochem.45:623-627 (1974)), T5 archaeal dna polymerase (Chatteriee etc., Gene 97:13-19 (1991)), PRD1 archaeal dna polymerase (Zhu and Ito, Biochim.Biophys.Acta.1219:267-276 (1994)) and T4 archaeal dna polymerase holoenzyme (Kaboord and Benkovic, Curr.Biol.5:149-157 (1995)). Utilize Φ 29 archaeal dna polymerases in the concrete embodiment.

Utilize the strand displacement factor, for example unwindase can promote strand displacement. Any archaeal dna polymerase that can carry out rolling-circle replication in the presence of the strand displacement factor is thought the described method that is applicable to, even this archaeal dna polymerase can not be carried out rolling circle amplification when not having this factor. The strand displacement factor that can be used for RCA includes but not limited to: the attached subunit of BMRF1 polymerase (accessory subunit) (Tsurumi etc., J.Virology 67:7648-7653 (1993)), adenovirus DNA-in conjunction with albumen (Zijderveld and van der Vliet, J.Virology 68:1158-1164 (1994)), hsv protein ICP8 (Boehmer and Lehman, J.Virology 67:711-715 (1993); Skaliter and Lehman, Proc.Natl.Acad.Sci.USA 91:10665-10669 (1994)), single-stranded DNA binding protein (SSB; Rigler and Romano, J.Biol. Chem.270:8910-8919 (1995)) and calf thymus unwindase (Siegel etc., J.Biol.Chem. 267:13629-13635 (1992)).

In other embodiments, this nucleic acid processing reaction is based on ligase dependence nucleic acid amplification. Specifically, United States Patent (USP) 4,883,750 have described the oligonucleotides joint test. The example of ligase dependent response is ligase chain reaction (LCR), wherein a kind of oligonucleotides and the first target sequence hybridization, the second target sequence hybridization that another kind of oligonucleotides and this first target sequence adjoin. The oligonucleotides of hybridization pairing is produced the connection product that contains two kinds of oligonucleotides with connecting substrate. If necessary, can replace (for example by sex change) from target sequence and connect product with other that produces this target sequence connecting product subsequently, so the oligonucleotides that is connected with this target nucleic acid complementation of having increased.

As shown in Figure 4, in the exemplary embodiment of the type, form the first crossbred by the first target sequence of mosaic type oligonucleotides and the first subsequence hybridization of target nucleic acid sequence, wherein 5 ' the nucleotides of 3 ' of this mosaic type oligonucleotides this thuja acid and this first subsequence is complementary. Then form the second crossbred by the second target sequence of the first collaborative oligonucleotides and the second subsequence hybridization of this target nucleic acid sequence, wherein this second subsequence is adjoined this first subsequence. Subsequently the 5 ' nucleotides of this first subsequence be connected reagent in the presence of few this thuja acid of this mosaic type is linked to each other with this first collaborative oligonucleotides, thereby formation contains the first and second subsequences of this target nucleic acid sequence and contains this mosaic type oligonucleotides and the first duplex of the connection product that is connected the first collaborative oligonucleotides. Those skilled in the art know suitable connection reagent available in these embodiments, include but not limited to: T4 dna ligase, e. coli dna ligase and thread hot bacterium (Tfi) dna ligase and their derivative of dwelling. In some embodiments, this connection reagent is heat endurance. After the connection, will connect product by sex change and separate with target nucleic acid sequence, thereby can make the second collaborative oligonucleotides form product with the mosaic type oligonucleotides that is connected product and the hybridization of the first collaborative Sequence. The second collaborative oligonucleotides has the blacked-out areas with the caught sequence complementation of mosaic type oligonucleotides, namely block sequence, this sequence is for the caught sequence and seizure oligonucleotide hybridization of blocking-up mosaic type oligonucleotides, thus so that signal oligonucleotides and seizure oligonucleotide hybridization. When the first collaborative oligonucleotides does not link to each other with the mosaic type oligonucleotides, when namely target nucleic acid sequence does not exist, the blacked-out areas of the second collaborative oligonucleotides usually not with can catch sequence hybridization, or only with lower affinity hybridization. It is known to those skilled in the art that can be so that blacked-out areas and the substance hybridization that can catch sequence occur when only having the first collaborative oligonucleotides to link to each other with the mosaic type oligonucleotides by following steps: size and/or (ii) complementarity of the caught sequence of this blacked-out areas and mosaic type oligonucleotides of (i) regulating the blacked-out areas of the second collaborative oligonucleotides; Or (iii) insert the incomplementarity sequence and make it to adjoin the blocking-up sequence.

The incomplementarity sequence base composition of blocking sequence is adjoined in adjustment makes it to be connected with first that the more or less ground of product is complementary can regulate in the following manner the sensitivity of detection system. If with incomplementarity sequence (B among Fig. 4₂And E₂Between section) be connected product with first and regulate (or " tuning ") for more complementary, with the sensitivity that increases detection system (namely, make it the lower target sequence of detectable concentration), if and it is lower for the complementarity that is connected product with first to regulate (or " tuning "), then the sensitivity of detection system. Therefore, can utilize tunable incomplementarity intervening sequence to regulate the sensitivity of processing reaction.

Fig. 5 A has shown another the exemplary example that adopts LCR, and it comprises the first target sequence (E with the first mosaic type oligonucleotides₁’-B ₁') hybridize to form the first crossbred with the first subsequence of target nucleic acid sequence, wherein 5 ' the nucleotides of 3 ' nucleotides of this first mosaic type oligonucleotides and this first subsequence is complementary, and the 5 ' nucleotides (this can catch the part that sequence is this first mosaic type oligonucleotides) that can catch sequence can not connect. Then make the second target sequence (E of the second mosaic type oligonucleotides₂’-B ₂') with target nucleic acid sequence in adjoin this first subsequence the second subsequence hybridize to form the second crossbred, the 3 ' nucleotides (this can catch the part that sequence is this second mosaic type oligonucleotides) that wherein can catch sequence can not connect. The 5 ' nucleotides of this first subsequence be connected reagent in the presence of, this the first mosaic type oligonucleotides is linked to each other with this second mosaic type oligonucleotides, comprise the first and second subsequences of this target nucleic acid sequence and contain simultaneously this first mosaic type oligonucleotides and the first duplex of the connection product that is connected the second mosaic type oligonucleotides thereby form. Those skilled in the art know suitable connection reagent available in this embodiment, include but not limited to: T4 dna ligase, e. coli dna ligase and thread hot bacterium (Tfi) dna ligase and their derivative of dwelling. In some embodiments, this connection reagent is heat endurance. This first duplex sex change is separated connect product and target nucleic acid. Can hybridize with seizure oligonucleotides (B among Fig. 5 A) by the connection product in this way, thus competition and signal oligonucleotides (B among Fig. 5 A ") combination. In this example, surpass the semaphore of preset range and the content inversely related of initial target sequence. In the improved form of this example, with the first mosaic type probe (E₁’-B ₁') and the second mosaic type probe (E₂’-B ₂') hybridization target sequence between " gap " (Fig. 5 B) arranged. After the hybridization, fill up E with polymerase first₁' and E₂' between the gap, use again ligase catalysis Connection Step. The perhaps gap filling oligonucleotides of the gap sequence complementation in adding and the target (E) in hybridization step, and this gap filling oligonucleotides participation coupled reaction.

Fig. 6 A has shown another the exemplary example that adopts LCR, comprise the first target sequence of the first mosaic type oligonucleotides and the first subsequence of target nucleic acid sequence (E) hybridized to form the first crossbred that wherein this first mosaic type oligonucleotides comprises the caught sequence (B of energy hybridization arrest oligonucleotides₃'), and the 5 ' nucleotides of 3 ' nucleotides of this first mosaic type oligonucleotides and this first subsequence is complementary. Make the second target sequence (E of the second mosaic type oligonucleotides₂’-B ₄') with target nucleic acid sequence (E) in adjoin this first subsequence the second subsequence hybridize to form the second crossbred, wherein this second mosaic type oligonucleotides comprise can the hybridization signal oligonucleotides the caught sequence (B of (B ")₄'). The 5 ' nucleotides of this first subsequence be connected reagent in the presence of, this the first mosaic type oligonucleotides is linked to each other with this second mosaic type oligonucleotides, comprise the first and second subsequences of this target nucleic acid sequence and contain simultaneously this first mosaic type oligonucleotides and the first duplex of the connection product that is connected the second mosaic type oligonucleotides thereby form. Then make this first duplex sex change come reaction product isolated and target nucleic acid. But by with substrate on the seizure oligonucleotides and the signal oligonucleotide hybridization that contains the test section come the detection reaction product, such as but not limited to latex bead, collaurum, enzyme, quantum dot (quantum dot) or signal amplification technique (SAT; For example United States Patent (USP) 5,902, and 724). In a rear example, the semaphore and the target sequence content in the primary sample that surpass preset range are proportional. Need to there be the gap sequence between the zone that polymerase is filled up and E1 ' and E2 ' are complementary in related embodiment (Fig. 6 B), thereby but or uses and have that suitable phosphorylation is terminal can carry out the joint gap fillibility oligonucleotides that enzymatic connects.

The connection product that ligase dependence embodiment of the present invention produces can also pass through RCA cyclisation and amplification.

Nucleic acid processing reaction of the present invention can comprise various other reagent that test can contain. These reagent comprise, such as salt, buffer solution, neutral protein matter (such as albumin), washing agent etc., and these reagent can be used for promoting best hybridization, chain is synthetic and detection, and/or reduce non-specific or the background interaction. According to the purity of sample preparation methods and target, also can use other reagent that improves test efficiency, such as protease inhibitors, nucleic acid inhibitor, antiseptic etc.

4. oligonucleotides

According to whether existing target nucleic acid sequence, the present invention to catch that oligonucleotides can be hybridized or " seizure " signal oligonucleotides or mosaic type oligonucleotides in the sample. In some embodiments, will catch oligonucleotides and be fixed on the surface of solids, and in other embodiments, it is free in the solution.

Be fixed in the embodiment on the surface of solids catching oligonucleotides, this surface can be made of the material natural, synthetic or natural generation of synthesis modification, includes but not limited to: cellulosic material, for example paper, cellulose and cellulose derivative are such as cellulose acetate; Glass or glass fibre; Natural or synthetic fabric; Plastics; Nylon; Porous gel, for example agarose; Silica gel, glucan and gelatin; Porous fibre matrix; Based on the material of starch, the crosslinked dextran chain of sephadex (Sephadex) for example; Ceramic masses; Latex; Polyvinyl chloride and PA membrane; Polystyrene; Merlon; And the combination of polyvinyl chloride-silica, etc.

In some embodiments, the surface of solids has formed the surface of carrying out processing reaction in the reaction vessel. In other embodiments, the surface of solids can also be to insert in the reaction vessel and the surface of the diagnosis band of taking out after the processing reaction of finishing for signal detection. In also having other embodiment, the surface of solids is optional mark the to assist particulate identified or the surface of pearl. In other embodiments, the surface is semiconductor nanowires (semiconducting nanowire), this surface should be configured to field-effect transistor, and at nucleotide sequence (for example, can catch sequence) after the seizure oligonucleotides that is fixed on nanowire surface is combined or is hybridized, can change conductance (referring to, such as Cui etc., 2001, Science 293:1289-1292; Hahm and Lieber, 2004, Nano Lett.4:51-54; Chen etc., 2003, Proc.Natl.Acad.Sci USA 100:4984-4989; Chen etc., 2004, J.Am.Chem. Soc.126:1563-1568 and Patolsky etc., 2004, Proc.Natl.Acad.Sci.USA 101:14017-14022).

Can adopt any suitable technology will catch oligonucleotides and be fixed on the surface of solids. For example, and Holstrom etc. (1993, Anal.Biochem.209:278-283) utilize biotin that the affinity of Avidin and Streptavidin is fixed in biotinylated nucleic acid molecules on the coated holder of Avidin/Streptavidin. Adoptable other method comprises uses poly--L-Lys or poly--L-Lys, Phe in advance coated polystyrene or glass solid phase, then utilize the covalently bound amino of bi-functional cross-linking agent or sulfhydryl modified oligonucleotides (Running etc., 1990, Biotechniques 8:276-277; Newton etc., 1993, Nucleic Acids Res.21:1155-1162). Kawai etc. (1993, Anal Biochem.209:63-69) have described connection short oligonucleotide probe to form polymer, it are cloned into the alternative of phagemid vector again. And then be connected on the XPS these oligonucleotides also fixing with 254nm UV irradiation. Also can directly be covalently attached to reference to the 5 '-phosphorylation Oligonucleolide primers that will lack the XPS (crosslinked (Covalink of chemical modification^TM) plate, Nan Ke company (Nunc)) method (Rasmussen etc., 1991, Anal.Biochem.198:138-142). Also can consider the article (1996 of O ' Connell-Maloney etc., TIBTECH 14:401-407), this piece article has disclosed respectively biotinylated oligonucleotide and sulfhydrylation oligonucleotides has been fixed in the coated silicon wafer of Streptavidin and the coated silicon wafer of iodoacetamide. Also amino modified oligonucleotides is fixed on the coated (Guo etc. on glass of isothiocyanates, 1994, Nucleic Acids Res.22:5456-5465) (Eggers etc. and on the coated wafer of epoxy silane (silane-epoxide), 1994, BioTechniques 17:516-5240). Said method is connected in substrate with Oligonucleolide primers after referring to synthesize. Perhaps, can adopt, for example the method original position synthetic oligonucleotide primer thing of (the same) such as Maskos and Southern (1992, Nucleic Acids Res, 20 1679-1684) or Fodor.

Those skilled in the art should know and can directly or indirectly be fixed on the surface of solids catching oligonucleotides. For example, can be adsorbed in the surface of solids with catching oligonucleotides, perhaps be covalently attached to spacer molecule, this spacer molecule is covalently attached to the surface of solids. Described spacer molecule can comprise emulsion particle, protein, for example bovine serum albumin(BSA) (BSA) or polymer, for example glucan or poly--(ethylene glycol). Perhaps, spacer molecule can comprise homotype-polynucleotides tail, for example oligomerization-dT.

Catch oligonucleotides and can be arranged in the surface of solids by any arrangement mode, as long as help detection signal oligonucleotides when the signal oligonucleotides is captured. In some embodiments, be arranged into the form that catches oligonucleotide arrays at the surface of solids with catching oligonucleotides. The ad-hoc location that will be fixed in corresponding to the seizure oligonucleotides of concrete target array helps to detect the result of multiple reaction. Be fixed in the embodiment of particulate or pearl will catching oligonucleotides, help to detect the result of multiple reaction according to the fixing seizure oligonucleotides corresponding to concrete target of the special ratios of the used particulate of reaction or pearl sum.

Seizure oligonucleotides of the present invention is not that target is specific usually, but manually can catch sequence specific (preferably) to contained each in mosaic type oligonucleotides described herein or the signal oligonucleotides, thereby can be with them as " general array ". That is, contain identical or limited group of array (solid phase or liquid phase array) that catches oligonucleotides. " liquid phase array " expression is passed through, for example the array in the solution of flow cytometry. In some embodiments, catch oligonucleotides and take the form on general surface; That is, comprise limited group of standard array that catches oligonucleotides making and to be applied to any purposes. This general array and the basically signal oligonucleotides coupling of complementation, signal oligonucleotides are combined with the seizure oligonucleotides in the presence of target sequence. Final user only need design the just customizable array of different mosaic type oligonucleotides that comprises any required target sequence, those skilled in the art will know that this is normally simple and cheap. Some embodiments have prepared the different artificial capture method oligonucleotide arrays that are generally; That is, catch oligonucleotides and known target sequence and do not have complementarity. Then can with the seizure oligonucleotides of array basically complementary caught sequence mix in the mosaic type oligonucleotides.

When having target nucleic acid sequence in the sample, signal oligonucleotides of the present invention and seizure oligonucleotide hybridization also provide at least part of detectable signal that shows this target nucleic acid sequence of existence in this sample. Therefore, the signal oligonucleotides comprise can with the nucleotide sequence that catches oligonucleotide hybridization and the signal reagent of being combined with this oligonucleotides.

Be combined with signal reagent in the signal oligonucleotides, comprise: (1) directly connects signal reagent and signal oligonucleotides; (2) indirect joint signal reagent and signal oligonucleotides (that is, connection signal reagent and subsequently the second intermediate of binding signal oligonucleotides); (3) the subsequent reactions product of connection signal oligonucleotides. Signal reagent should be directly connected in the signal oligonucleotides. Signal reagent can be selected from: chromogen, catalyst, enzyme, fluorogen, light emitting molecule, chemiluminescent molecule, lanthanide ion are (such as europium (Eu³⁴)), radio isotope and direct visible signal reagent. Direct visible signal reagent can utilize colloidal metal or non-metallic particle, dye granule, enzyme or substrate, organic polymer, latex particle, liposome, branch aggressiveness (dendrimer) (or branch aggressiveness sample or jugate (concatenated) nucleic acid structure, SAT for example) or contain other carrier that produces semiochemicals, etc. U.S.4,366,241; U.S. 4,843,000 and U.S.4,849,338 a large amount of applicable enzymes of making signal reagent have been disclosed. Can be used for suitable enzymes signal reagent of the present invention and comprise alkaline phosphatase, horseradish peroxidase, luciferase, beta galactosidase, glucose oxidase, lysozyme, malic dehydrogenase etc. Can use separately the enzyme signal reagent, or with solution in the second enzyme coupling. Perhaps, the fluorogen that can be used as appropriate signals reagent of the present invention includes but not limited to: fluorescein, rhodamine, texas Red, fluorescein or R-PE. Will be appreciated that also in the situation of indirect joint signal reagent (2) that reagent such as biotin, digitophyllin, Streptavidin and range protein antigen can be used as joint and needs to exist the second intermediate to produce detectable signal. For biotin, the second intermediate can comprise the Streptavidin enzyme conjugates. For antigen signals reagent, the second intermediate can comprise antibody-enzyme conjugates.

Be free in some embodiment in the solution catching oligonucleotides, catch the part that oligonucleotides comprises signal detection system, the signal oligonucleotides comprises another part of this signal detection system, and wherein each several part cooperates to produce and shows the first signal that has target nucleic acid sequence in the sample or show the secondary signal that does not have target sequence in the sample. In exemplary example, catch oligonucleotides and comprise acceptor fluorescence group, the signal oligonucleotides comprises the donor fluorogen, thus when the signal oligonucleotides when catching oligonucleotide hybridization, acceptor and donor fluorogen are enough approaching and then bring out FRET (FRET). Detect FRET and can provide the signal that has target nucleic acid sequence in the sample, can provide the signal that does not have this sequence in the sample and lack FRET. The technical staff knows, at least a detection FRET in available following two kinds of methods: fluorescence method or quench method. In fluorescence method, fluorescence detector is arranged at the emission spectrum of acceptor fluorescence group, donor shifts and the fluorescence of acceptor shows that the signal oligonucleotides is combined with the seizure oligonucleotides to the energy of acceptor. In quench method, detector is arranged at the emission spectrum of donor fluorogen, donor shifts and the quencher of donor spectrum shows that the signal oligonucleotides is combined with the seizure oligonucleotides to the energy of acceptor.

Mosaic type oligonucleotides of the present invention comprise with the first target sequence of the subsequence of target nucleic acid sequence hybridization and with the caught sequence that is selected from the sequence hybridization that catches oligonucleotides or signal oligonucleotides. As mentioned above, when not having target nucleic acid sequence, the caught sequence of this mosaic type oligonucleotides and seizure oligonucleotide hybridization and disabling signal transduction. In some embodiments, the caught sequence of this mosaic type oligonucleotides preferentially with catch oligonucleotide hybridization. This may be because the mosaic type oligonucleotides concentration that exists is higher than due to the signal oligonucleotides, but perhaps modified chimeric type oligonucleotides, thus make itself and the affinity that catches oligonucleotide hybridization be higher than the signal oligonucleotides.

For target sequence, can catch sequence normally non-natural (that is, exogenous) nucleic acid, but add this target sequence or be attached thereto. Should be noted that in this application " target sequence " can comprise the primary sample target sequence, or the target of deriving, for example reactant of reaction described herein or product; Therefore this target sequence can be the probe, PCR product of the first linking probe in the OLA reaction or connection etc. Can catch sequence as the unique identifier (identifier) of mosaic type oligonucleotides, thereby become the unique identifier of target sequence. In a word, can, for example on the array many groups of exploitations can catch sequence and corresponding catch oligonucleotides with at utmost reduce them to each other and with reactant mixture in other component, comprise that crisscrossing occurs the sequence (for example, the sequence in the genomic DNA) beyond the target sequence in target sequence and the larger nucleic acid sequence. U. S. application publication No. 20030096239 has disclosed some and can catch or " adapter (adapter) " sequence. The exemplary sequence that catches is the sequence that meets following standard. They do not find that they do not have harmful structure, for example hairpin loop in genome (preferred human genome).

In addition, according to the structure of system, those skilled in the art will know that and to catch sequence and mix 3 ' or 5 ' end of mosaic type oligonucleotides, or interior location.

Those skilled in the art will know that the length that can catch sequence can be because of required combination " intensity " and the required different sequence quantity differences that catch. In some embodiments, can catch sequence and generally be about about 500 base-pairs of 6-, normally about 8-is about 100, and more common is about 25 base-pairs of about 10-.

In some embodiments, can catch the unique target sequence that to be combined with the target sequence that identifies of sequence. That is, need not to be combined with target sequence although can catch sequence itself, whether this system can catch sequence and detect target sequence by detecting to exist. Therefore, will be used as the sign that has target sequence to the detection that can catch sequence subsequently.

The technical staff of biology field knows method and the algorithm of the suitable subsequence of identifying target nucleic acid sequence, and these methods and algorithm are widely used. These methods comprise sequence alignment method (Gotoh O.Multiple sequence alignment:algorithms and applications. " sequence alignment: algorithm and application " Adv Biophys.1999; 36:159-206. Lecompte O, Thompson JD, Plewniak F, Thierry J, Poch O./Multiple alignment of complete sequences (MACS) in the post-genomic era. " the full length sequence multiple ratio of genome times afterwards comprehensively is to (MACS) " Gene.2001 May 30; 270 (1-2): 17-30), dot matrix method and database index method, for example basic gopher of local sequence alignment and FASTA program (Pearson WR.Flexible sequence similarity searching with the FASTA3 program package. " the flexible sequence similarity retrieval of employing FASTA3 program package " Methods MoI Biol.2000; 132:185-219; Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) " Basic local alignment search tool. " " the basic gopher of local sequence alignment " J.MoI.Biol.215:403-410). The subsequence of target nucleic acid sequence can be the conservative region in the nucleotide sequence family, perhaps can be zone unique in the concrete target nucleic acid sequence.

The method of target sequence that design can be hybridized the subsequence of target nucleic acid sequence also is to know and widely used.In this, can be with reference to Dieffenbach etc., 1995 " PCR primers: laboratory manual " (PCR Primer:A Laboratory Manual), CSHL press (CSHL press), cold spring port, the U.S.; Erlich, 1989, " round pcr: the principle of DNA cloning and application " (PCR Technology:Principles andApplications for DNA Amplification), Stockton press (Stockton Press), New York; Innis etc., 1990, " PCR scheme: method and application guide " (PCR Protocols:A Guide toMethods and Applications), academic press (Academic Press), New York; Ellingboe etc., 1992, " round pcr: dna sequencing " (The PCR Technique:DNA Sequencing), Eton publishing company (Eaton Publishing Co.), Nei Dike, Massachusetts; Bej etc., 1991, " biological chemistry and molecular biological important summary " (Critical Reviews in Biochemistry and MolecularBiology), 26:301-334; Hughes etc., 2001, Nat.Biotechnol.19:342-347; Kane etc., 2000, Nucleic Acids Res.28:4552-4557; Relogio etc., 2002, Nucleic AcidsRes.30:e51; Wang etc., 2003, Bioinformatics 19:796-802; Chen etc., 2002, BMCBioinformatics 3:27; Hill etc., 2002, information biology and Chemoinformatics target summary (Proceedings of Objects in Bio-﹠amp; Chem-Informatics), OMG management by objectives group (OMGObject Management Group), Washington, district of Columbia (U.S.), 39-44 page or leaf; Li etc., 2001, Bioinformatics 17:1067-1076; Lipshutz etc., 1999, Nat.Genet.21:20-24; Nielsen etc., 2003, Nucleic Acids Res.31:3491-3496; Raddatz etc., 2001, Bioinformatics 17:98-99; Rahmann, 2003, Journal of Bioinformatics andComputational Biology 1:343-361; Reymond etc., 2004, Bioinformatics 20:271-273; Rimour etc., 2003, the healthy grid conference summary (Proceedings of the firstHealthgrid Conference) of the first, Lyons, France, 88-96 page or leaf; Rouillard etc., 2002, Bioinformatics 18:486-487; Rouillard etc., 2003, Nucleic Acids Res.31:3057-3062.

Target sequence subsequence common and target nucleic acid sequence is complementary basically.For example, if the subsequence of target nucleic acid sequence is A-G-T-A-C-T-G, then complementary target sequence can be T-C-A-T-G-A-C.Calculate the subsequence of target nucleic acid sequence and the characteristic of their complementary sequence, and be used to optimize the design of target sequence.The characteristic of being paid close attention to includes but not limited to: oligonucleotide length (in the base residue), T _mWith the proneness of self hybridizing.Those skilled in the art know the algorithm of these characteristics of assessment, these algorithm widespread uses.Can be with reference to Jarman, 2004, Bioinformatics 20 (10): 1644-1645; Gibbs etc., 1997, J.Virol.Methods63 (1-2): 9-16; Gibbs etc., 1998, J.Virol.Methods, 74 (1): 67-76; Antoniw, 1995, Mol Biotechnol 4 (2): 111-119; Le Guyader etc., 1996, Arch.Virol.141 (11): 2225-2235; Chen etc., 1995, Virus Res.39 (2-3): 365-375; Wilson etc., 1993, J.Clin.Microbiol.323:1573-1580; Chen etc., 1989, FEMS Microbiol.Lett.57:19-24; Greigen etc., 1994, J.Clin.Microbiol.32:335-351; Evertsson etc., 2000, APMIS108:385-392; Hryniewiecki etc., 2002, J.Heart Valve Dis.11:870-874; Rothman etc., 2002, J.Infect.Dis.186:1677-1681; McCabe etc., 1995, Pediatrics 95:165-169; Lisby etc., 2002, Infect.Dis.Clin.North Am.16:393-412; Ley, 1998, Eur.J.Clin.Microbiol.Infect.Dis.17:247-253.

Interoperability oligonucleotide of the present invention comprises and the second target sequence that is selected from following sequence hybridization: the different subsequence of subsequence that is hybridized with the first target sequence in the target nucleic acid sequence; Or the subsequence of the complementary strand of target nucleic acid sequence; Or at least one mosaic type oligonucleotide.In the nucleic acid processing reaction, when having target nucleic acid sequence to exist, the interoperability oligonucleotide is cooperated with the mosaic type oligonucleotide and is formed reaction product.In the present invention in full, reaction product comprises first chain and second chain, first chain comprises at least a mosaic type oligonucleotide or its extension products, second chain comprises at least a interoperability oligonucleotide or its extension products, thereby the caught sequence of having blocked the mosaic type oligonucleotide with catch oligonucleotide hybridization, and then make the signal oligonucleotide with catch oligonucleotide hybridization.

Can adopt the used oligonucleotide of prepared by any suitable process the inventive method, for example (1979, Methods Enzymol.68:90) and United States Patent (USP) 4,356,270 described phosphotriester methods such as Narang.Perhaps, can adopt (1979, Methods Enzymol.68:109) described phosphodiester methods such as Brown to carry out this preparation.Also can adopt the automatic embodiment of above method.For example, in a kind of so automatic embodiment, with the diethyl phosphoramidite as initial substance, and as Beaucage etc., (1981, Tetrahedron Letters 22:1859-1862) described synthesizing.Also can be with reference to United States Patent (USP) 4,458,066 and 4,500,707, these two pieces of patents relate to the method for synthetic oligonucleotide on the modified solid upholder.Also may use from the isolating oligonucleotide of biological origin (for example, the denatured strand of the restriction endonuclease digestion product of plasmid or phage DNA).In some embodiments, according to United States Patent (USP) 5,424, the method synthetic oligonucleotide that 186 (Fodor etc.) are disclosed.This method adopts photolithographic techniques at the synthetic multiple different oligonucleotide of the accurate known location of substrate surface.Perhaps, can adopt the method for PCR-based, for example (2000, Nucleic AcidResearch 28 (12): e58) described method produces oligonucleotide for Antson etc.The method of these PCR-based is specially adapted to produce longer, particularly is longer than the oligonucleotide of 100 Nucleotide.

Be suitable for that oligonucleotide is hybridized each other or with the condition of target nucleic acid (comprising DNA or RNA) hybridization under according to the inventive method hybrid nucleic acid.In this, can reference, for example NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH (" nucleic acid hybridization, a kind of practical approach ") (Homes and Higgins compile) (IRL press (IRL press), Washington, the district of Columbia, 1985).Whether hybridization the influence of following factor takes place to be subjected to usually: the concentration of length nucleic acid, pH, temperature, monovalence and divalent cation, the crossbred of nucleic acid form ratio, the dielectric viscosity of G and C in the zone and may have denaturing agent.This variable also influences the required time of hybridization.Therefore, optimum condition depends on concrete application.Yet, but these empirical condition of conventional determining and need not too much experiment.

In some embodiments, the inventive method adopts the high hybridization conditions of distinguishing.For example, can be with reference to Wallace etc. (1979, Nucl.Acids Res.6:3543), they have described with containing a unmatched similar oligonucleotide probe of inner base pair and have compared, and difference is the condition of coupling and the oligonucleotide probe hybridization situation grown with the complete homologous 11-17 of target sequence base fully.Also can be with reference to Wood etc. (1985, Proc.Natl.Acid.Sci USA 82:1585), they have described the hybridization conditions of 11-20 the base long oligonucleotide that utilizes the 3M tetramethyl ammonium chloride, and wherein the melting temperature(Tm) of crossbred only depends on the length of oligonucleotide probe and its GC content no matter.In addition, Drmanac etc. (the same) have described 6-10 the hybridization conditions that the long oligomer of Nucleotide carries out strict hybridization, utilize nucleotide analog, for example ' locking ' (locked) nucleic acid be easy to obtain similar condition (Christensen etc., 2001 Biochem J 354:481-4) most.

Hybridization can contain hybridization and optimizes agent usually optional, for example waits under the hybridization buffer existence of stablizer (isostabilizingagent), denaturing agent and/or renaturation accelerator and carries out.Example Deng stablizer includes but not limited to: trimethyl-glycine and rudimentary tetraalkylammonium salt.Denaturing agent is the composition that reduces the double chain acid molecule melting temperature(Tm) by the hydration of hydrogen bond between the base or nucleic acid molecule in the interference double-strandednucleic acid.Denaturing agent includes but not limited to: methane amide, formaldehyde, dimethyl sulfoxide (DMSO), acetate tetra-ethyl ester, urea, guanidinium isothiocyanate, glycerine and chaotropic salt.The hybridization accelerator comprises hnRNP (hnRP) A1 and cationic detergent, for example CETRIMIDE POWDER (CTAB) and bromination dodecyl TMA (TriMethylAmine) (DTAB), polylysine, spermine, spermidine, single strand binding protein (SSB), phage T4 gene 32 albumen and ammonium acetate and alcoholic acid mixture.

It is also understood that if nucleotide sequence of the present invention (for example contains two chains, genomic dna) or have and (for example to hinder oligonucleotide hybridization and/or extension, RNA) secondary structure, chain independent or separated nucleic acid sequence in synthetic extension primer molecule is an ideal.Can adopt any suitable denaturation method, for example physics, chemistry or Enzymology method realize that this chain separates.A kind of physical method that separates each chain of polynucleotide sequence comprises that this nucleotide sequence of heating is until its (＞99%) sex change fully basically.Conventional thermally denature can be included in about 80 ℃-105 ℃ temperature and carry out 1-10 minute.Also can utilize certain enzyme in the enzyme classes that is called helicase or enzyme RecA (having helicase activity), have the known ribose ATP (riboATP) that can make the DNA sex change to induce chain to separate in the presence of (rATP).Kuhn Hoffmann-Berling (1978, CSH-Quantitative Biology 43 63) suitable reaction conditions of using each chain of helicase isolating nucleic acid has been described, Radding (1982, Ann.Rev.Genetics 16 405-437) has summed up the technology of utilizing RecA.Perhaps, can pass through, for example apply and make low tension carry out electric sex change (Purvis etc. by sample, 1996, the 4th international biosensor conference (4th World Congress onBiosensors), Bangkok, the 39th page, Ai Ersaiwei High-tech company (Elsevier AdvancedTechnology), the Oxford, this part document is included this paper in as a reference), or (for example handle sample with diluted acid, handle 7.5-10 minute (Meinkoth and Wahl, 1984, Anal Biochem.138267-284) with 0.25 M HCl.

5. detect

The character of basis signal reagent can be by the detectable signal of range estimation or instrumental method detection signal oligonucleotide of the present invention.By detecting fluorescence with the rayed fluorescent mark and with fluorophotometer; By providing enzyme system to produce the dyestuff that available spectrophotometer detects; Or utilize reflexometer to detect dye granule or colored colloidal metal or non-metallic particle; In the situation of utilizing radio-labeling or chemiluminescent molecule, come the instrument detecting signal with radiation counter or radioautography.Therefore, available proofing unit detection or the scanning light relevant with mark, described light can comprise fluorescence, luminous, focused beam or laser.In this case, can utilize charge coupled device (chargecouple device) (CCD) or photocell come the light of the seizure oligonucleotide of each position in the scanning array/signal oligonucleotide hybridization body emission, and with the direct record data of digital machine.Perhaps, be fixed on particulate or the pearl then available fluorescent activation cell sorting (FASC) technology for detection signal reagent if catch oligonucleotide.In some cases, the instrument detecting signal is optional.For example, as described herein, the color spot that utilizes glue state metal particle relevant or enzymatic to produce with array format (format), the range estimation array can be understood the pattern of array.

In some embodiments, fixing solid surface of catching oligonucleotide also can change into plain language heredity distribution (plain language geneticprofile) with signal mode from (for example) array with pattern (pattern) identification equipment and software coupling.In some embodiments, utilize the signal that signal reagent produces on " chip reader " detection arrays.For example, Pirrung etc. (United States Patent (USP) 5,143,854) have described " chip reader " available detection system.The chip reader also can determine that the signal of concrete array position or element is true positives or may is spurious response in conjunction with some signal processing (method) usually.For example, Fodor etc. (United States Patent (USP) 5,925,525) have described exemplary chip reader.

In concrete embodiment, according to the content of the signal oligonucleotide of catching oligonucleotide hybridization, detectable signal of the present invention not only shows the existence of target nucleic acid sequence, also shows the content of target nucleic acid sequence.In this, quantitative assay target nucleic acid sequence by any method known to those skilled in the art is such as but not limited to the known standard nucleic acid sample of reference concentration.

Also can help understand detectable signal of the present invention by including the positive and negative control in.Those skilled in the art are not difficult to determine suitable contrast.Positive control can comprise the mosaic type oligonucleotide, comprises the sequence (for example beta-actin sequence) that has existing in the sample in this mosaic type oligonucleotide and has specific first subsequence.Negative control can comprise the mosaic type oligonucleotide, and this mosaic type oligonucleotide lacks the first target specificity subsequence or the target specificity subsequence that had does not hybridize with target nucleic acid sequence basically.

6. sample

The suitable sample of available of the present invention comprises from any biological strand or double-strandednucleic acid extract that obtains, or their copy.Extract can obtain from any source of biology, such as but not limited to: lysate, cell, organize or be derived from other material of virus, fungi, bacterium, plant and animal.

Can prepare the sample extraction thing (for example DNA or RNA) of nucleic acid according to the lysis step, described step includes but not limited to: handle with SDS, osmotic shock, ultrasonication, guanidinium isothiocyanate and N,O-Diacetylmuramidase and realize cracking.The suitable DNA of available of the present invention comprises genomic dna or cDNA.Can be by " up-to-date molecular biology method " (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) for example volumes such as () Ausubel ((John Wiley ﹠amp of John Willie father and son company limited; Sons, Inc.), 1995) any and in the multiple conventional method of using described of " molecular cloning laboratory manual " (MOLECULAR CLONING.A LABORATORY MANUAL) volumes such as () Sambrook (cold spring port press (Cold Spring Harbor Press), 1989) prepares this DNA.Can (1987, Anal.Biochem.162:156) any appropriate method of Miao Shuing prepares RNA sample extraction thing by for example " up-to-date molecular biology method " (the same), " molecular cloning laboratory manual " (the same) and Chomczynski and Sacchi.

Those skilled in the art should know, the sample extraction thing can comprise any amount of things, includes but not limited to: body fluid (including but not limited to blood, urine, serum, lymph, saliva, anus and vaginal secretions, sweat and the seminal fluid of in fact any biology); Environmental sample (including but not limited to air, agricultural, water and soil earth sample); The biological warfare agent sample; Study sample; The sample of purifying, for example genomic dna of purifying, RNA, protein etc.; Primary sample (bacterium, virus, genomic dna etc.).Sample also can comprise sample mixture, and for example two or more sample mix of different sources together.Those skilled in the art should know, in fact can carry out any experimental implementation to sample.

7. reaction formation

Can comprise and implement analytical procedure of the present invention in test tube, micropore, wafer (that is chip) and the microfluidic devices at any suitable reaction vessel.In some embodiments, with at least a manufacturing or the pre-container made of catching in oligonucleotide, mosaic type oligonucleotide, interoperability oligonucleotide, signal oligonucleotide, reaction buffer, enzyme, signal reagent and the nucleotide precursor.Optimized the reactive component of content in advance and preferably be included in one or more reaction vessels for implementing analytical procedure of the present invention with solution or lyophilized form.In the exemplary example of the type, the final user will measure with enforcement in this reaction vessel of at least a adding in nucleic acid samples, nucleic acid processive enzyme, mosaic type oligonucleotide and the interoperability oligonucleotide simply.

In some embodiments, reaction vessel is placed controlled thermal (thermocontrollable) environment (for example thermal cycler, incubator etc.) carry out each step of analytical procedure of the present invention.In other embodiments, described reaction vessel can be carried out a step used in these analytical procedures or multistep operation.These operations include but not limited to: mix; Filter; Nucleic acid extraction; Nucleic acid purification; In conjunction with; Wash-out; For hybridize, nucleic acid processing reaction (for example, PCR, LCR, OLA, RCR etc.) and make the nucleic acid hybrids sex change and control heat; With the detection signal oligonucleotide.For example, U.S. Patent Application Publication 2002/0115200,2002/0173032,2003/0008286 and 2005/0142565 has disclosed the exemplary device of the type.

8. test kit

Can will be contained in the test kit together according to all required basal components of target nucleic acid sequence in the inventive method analytical sample.These test kits are optional to be equipped with the suitable ingredients, the positive and the negative control that make visibleization of observation signal reagent, dilution buffer liquid etc.The component that is fit to nucleic acid is implemented the nucleic acid processing reaction also can be housed.These components comprise various polysaccharases, such as but not limited to: (depending on the nucleic acid processing reaction technology that is adopted) such as Tag polysaccharase, reversed transcriptive enzyme, dna ligases, nucleotide precursor and buffering solution.This test kit also can be equipped with the different vessels that is used for each component.In some embodiments, these test kits are equipped with the working instructions of implementing the inventive method.

In embodiment, as described in the embodiment of chapters and sections 7, comprise in the test kit of the present invention the reaction vessel that is fixed with the seizure oligonucleotide and contains previously prepared reagent mixture (preferred lyophilized form) is housed.

For making the present invention be convenient to understand and enforcement, particularly preferred embodiment has been described by following non-limiting example.

Embodiment

Embodiment 1

RT-PCR detects influenza A virus pol gene section PB2

Design of primers:

The increase part of influenza A virus PB2 section of design RT-PCR primer.Use inosine in many sites or mix base, thus the pol gene of many different virus that can increase.One of primer is a mosaic type, can catch sequence or " label " as long as 5 ' end of PCR primer contains.In the present embodiment used caught sequence be 5 '-CTT TAA TCT CAA TCA ATA CAA ATC-3 ' ([SEQ ID NO:1] is labeled as B ' in Fig. 2), its be design (thinking) as United States Patent (USP) 6,027,884 described sequences.Can use in target sequence (influenza A virus) undiscovered any other can catch sequence.

Mosaic type influenza virus forward primer Ch-PB2 (1) F contain sequence 5 '-CTT TAA TCT CAA TCAATA CAA ATC AG (C/T) TCI TC (C/T) TT (C/T) AG (C/T) TT (C/T) GG-3 ' [SEQ IDNO:2].Influenza virus reverse primer PB2 (2) R contain sequence 5 '-AGT AT (T/C) CTC AT (T/C) CC (T/A) GAN CC-3 ' [SEQ ID NO:3].The expection size of product that these primers produce is about 1010bp.Yet this may be different according to the virus that exists in the interested sample, because the length of PB2 encoding sequence slightly different (Fig. 1) between the isolate.

From sample, extract RNA:

According to manufacturer's working instructions, utilize the easy extraction test kit of QIAGEN RNA to extract the viral RNA of sample (for example, amniotic fluid or clinical sample).Add 600 μ L guanidine denaturing agents and 6 μ L2-mercaptoethanols earlier with deactivation 100 μ L samples, use the QIAGEN extraction scheme again.The RNA that extracts is resuspended in the water that 50 μ L do not contain the RNA enzyme (proper root, QIAGEN).The resuspended RNA of 2 μ L is used for the RT-PCR reaction.

Quantitative assay:

With about 100 picomole catch oligonucleotide (being labeled as B among Fig. 2) (spacer groups-5 '-GAT TTG TATTGA TTG AGA TTA AAG-3 '; [SEQ ID NO:4]) be combined in shape and be applicable on each hole of the band of test tube of thermal cycling machine or 96-orifice plate.Can utilize 5 ' modification group (amino that for example, between 5 ' end of amino and seizure oligonucleotide, contains (CH2) 6 " at interval " sequence (or similar sequence)) realization seizure oligonucleotide to combine with matrix.Perhaps, can will be able to be mixed an end of catching oligonucleotide by 5 ' biotin moiety of flat board in conjunction with the Streptavidin bag so that high avidity is non-covalent.

With the signal oligonucleotide be designed to contain with the test section (for example, latex beads) link coupled sequence 5 '-CTTTAA TCT CAA TCA ATA CAA ATC-3 ' ([SEQ ID NO:1], be labeled as B ") among Fig. 2, it is added with catching each hole that oligonucleotide wraps the microwell plate of quilt in advance.The add-on of signal oligonucleotide and fixed are caught the oligonucleotide amount about equally.Can come the accurate amount of definite seizure oligonucleotide that will add by rule of thumb by each test of titration and each batch reagent.

The standard substance of quantitative assay:

986bp section with the PB2 gene is cloned among the plasmid PCR TOPO2.1 (hero company (Invitrogen)).The virus sequence complementary portion complementation of sequence of this plasmid (this paper is called " control plasmid ") and forward primer Ch-PB2 (1) F and reverse primer PB2 (2) R.The serial dilution that can utilize control plasmid is as the quantitative assay standard substance of test and the reagent contrast of RT-PCR reaction.With every hole 0-10, the plasmid serial dilution of 000 picomole can be used as quantitative assay (standard substance) and reagent contrast.When fixing about 100 picomole in every hole are caught primer, the concentration range of quantitative assay standard substance should be every hole 0-10 nmole, comprise every

hole

0,3,10,30,100,300,1000,3000 and 10,000 picomole, every kind of concentration of standard substance is used the same form two holes.

The RT-PCR reaction:

1. as follows, to the thermal cycler programming, synthesize thereby can behind pcr amplification, carry out cDNA immediately:

CDNA is synthetic:

1 takes turns: 46 ℃ added in 30 minutes 60 ℃ 10 minutes

Sex change:

1 takes turns: 94 ℃ 2 minutes

Pcr amplification

8 take turns cooling (step down) PCR that is made of following steps:

94 ℃ of sex change 15 seconds

Take turns in the successive " cooling round " at each, 56 ℃, 54 ℃, 52 ℃, 50 ℃, 48 ℃, 46 ℃, 44 ℃, 42 ℃ annealing 30 seconds

68 ℃ were extended 75 seconds

Carry out then:

36 take turns: 94 ℃ 15 seconds (sex change)

40 ℃ 30 seconds (annealing)

68 ℃ 75 seconds (extension)

The final extension (optional)

1 takes turns: 68 ℃ 5 minutes

2. the thin-walled PCR test tube (Qi Bishang combines in advance and catches oligonucleotide) that on ice following material adding 0.2mL is not contained nuclease:

2 * reaction mixture, 25 μ L

The serial dilution (as the quantitative assay standard substance) (2 μ L) of template ribonucleic acid 2 μ L or control plasmid

Adopted primer Ch-PB2 (1) F2 μ L (160 picomole) is arranged

Antisense primer 2 μ L (160 picomole)

Superscript III RT/ platinum label mixture (Superscript III RT/PlatinumTaq Mix) (hero company) 2 μ L

2 μ L signal oligonucleotide (100 picomole or other content measured in advance by titration)

Add to 50 μ L with the water that does not contain the RNA enzyme.

3. soft mixing is also simple centrifugal to guarantee that all components is positioned at the bottom of amplification test tube.Reaction plate or test tube are placed the thermal cycler that preheats of programming as mentioned above.

4. along with reaction is carried out, mosaic type influenza virus forward primer Ch-PB2 (1) F5 '-CTT TAA TCTCAA TCA ATA CAA ATC AG (C/T) TCI TC (C/T) TT (C/T) AG (C/T) TT (C/T) GG-3 ' [SEQ ID NO:2] mixes double-stranded PCR product and is isolated, thus its can not with fixed catch oligonucleotide (spacer-5 '-GAT TTG TAT TGA TTG AGA TTA AAG-3 '; [SEQ ID NO.4]) and the signal oligonucleotide between in conjunction with competing.

Therefore, the signal oligonucleotide binding capacity in " annealing " stage of each PCR round carries out with reaction and increases.

To contain in the test tube/hole of the unknown (material) strength of signal with in order to obtain typical curve admixture the strength of signal in the test tube/hole of plasmid standard of serial dilution make comparisons.By comparing with typical curve, the concentration of the virus sequence that can calculation sample hits.

And nonessentially produce typical curve with the continuous test tube/hole of discrete.In one situation of back, can use " interior mark ", wherein can use the different mosaic type primers and the pairing of signal oligonucleotide of an examination criteria RNA or plasmid, irrelevant standard rna of content known sequences or plasmid are increased jointly with unknown sample in same reaction tube.The signal that mark RNA or plasmid produce in then in semaphore that unknown sample can be produced and the same test tube is made comparisons.

Detection signal strength in the time of can finishing in whole rounds of PCR reaction perhaps detects every strength of signal of taking turns the PCR reaction with reading device.

Perhaps, in another embodiment, the signal oligonucleotide can be mixed (for example, by freeze-drying) reaction tube in advance together with all the components beyond the removing template RNA.Can rebuild reaction mixture by adding water.

Embodiment 2

" terminal point " detects

Present embodiment adopts " terminal point " detection step isolated with amplification step.As described in embodiment 1, detection reagent and signal reagent are added in 96 orifice plates, wherein every hole 100 picomole of fixedly having an appointment are caught oligonucleotide.Can catch mosaic type RT-PCR primer (Ch-PB2 (1) F5 '-CTT TAA TCT CAATCA ATA CAA ATC AG (C/T)-TCI TC (C/T) TT (C/T) AG (C/T) TT (C/T) GG-3 ') [SEQID NO:2] shown in embodiment 1.The signal oligonucleotide is shown in embodiment 1, and through biotinylation, its amount in detecting the hole equates (though this will carry out titration to various analyte) with the mole number of catching oligonucleotide.Used those of RT-PCR primer and embodiment 1 are identical.

The RT-PCR reaction is carried out as described in embodiment 1, does not catch oligonucleotide and signal oligonucleotide but do not comprise during the RT-PCR.The plasmid contrast of including serial dilution in is as the quantitative assay standard substance.After RT-PCR reacts end, reaction product 94 ℃ of heating 5 minutes, then with wet ice quencher, and is transferred to the detection flat board.

Along with the PCR reaction is carried out, mosaic type can be caught the PCR primer and be mixed in the double-stranded product, and it is lower with the efficient of catching competitive oligonucleotide binding signal oligonucleotide.The content of PCR product was high more when reaction finished, and is just weak more to the competition of binding signal oligonucleotide.

37 ℃ leave standstill 20 minutes after, 1 * SSC pH7.2 washing with every hole 300 μ L detects dull and stereotyped three times, 1: 10 of the Streptavidin superoxide enzyme conjugates (Luo Shi catalog number (Cat.No.) 1089153) that PBS/0.1% tween/0.5%BSA is prepared then, 000 diluent adds in the hand-hole, cultivated 30 minutes for 37 ℃, subsequently with PBS/ tween washing three times.Upset drains each flat board, adds substrate again in each hole.Add with 100mM sodium acetate/citric acid then, 0.42mM TMB (Luo Shi catalog number (Cat.No.) 11484281001), the 0.004%H2O2 (v/v) of pH4.9 preparation.Use 2M H ₂SO ₄Termination reaction.Formed product at first is blue, is yellow behind the reaction terminating, water soluble.With 650nm is reference wavelength, detects the 450nm absorbancy with the dull and stereotyped reader of ELISA.

Embodiment 3

The ligase enzyme dependent response

The used DNA of this one exemplary embodiment is control plasmid DNA, and it contains the double-stranded DNA copy of influenza A virus PB2 constant gene segment C coding region.In this plasmid, the 986bp section of PB2 gene has inserted among the plasmid PCR TOPO2.1 (hero company).

Mosaic type connects oligonucleotide sequence:

With attachable oligonucleotide be designed to influenza A virus in comprise the regional complementarity of the codon 627 digital site of site equivalence (or in the PB2 sequence of influenza A virus).Ch-PB2U 5 '-CTT TAA TCTCAA TCA ATA CAA ATC TTG CAG CIG CIC CAC CIG-3 ' ([SEQ ID NO:5] is labeled as B '-E1 ' among Fig. 4).PB2D-5 '-AIC AIA GIA GIA TGC AGT-3 ' ([SEQ ID NO:6] is labeled as E2 ' among Fig. 4).It is identical that the upstream mosaic type connects the used sequence of caught sequence used in the oligonucleotide and embodiment 1.

For exemplary purpose, utilize above-mentioned standard plasmid DNA as target, use heat-staple dna ligase to carry out the ligase enzyme dependent response.Process (the Belgrader etc. as described below of the PB2 section part of influenza A virus in amplification and the detection control plasmid (above-mentioned), 1995, Genetic Identity ConferenceProceedings (" genetic identity conference summary "), the 6th human evaluation international symposium Sixth InternationalSymposium on Human Identification improves one's methods):

5 μ L equal portions plasmids are diluted in the 20 μ L LCR mixtures, contain 50mMTris/HCl pH8.5 in this LCR mixture, 50mM KCl, 10mM MgCl ₂, 1mM NAD ⁺, 10mM DTT, the Taq dna ligase of LCR oligonucleotide group (each primer of 50 picomole, Ch-PB2U and PB2D) and 10 units.Thermal cycling is following to be carried out: carry out 1 earlier and take turns 95 ℃ of sex change of 2 minutes, carry out 95 ℃ of sex change of 30 seconds of 20-25 wheel and 65 ℃ of 4 minutes are connected again.

The reaction vessel that carries out ligase enzyme mediated responses step is integrated with the detection system that comprises custom-designed second kind of interoperability oligonucleotide (being labeled as B2-E2 in Fig. 4).

For detection system, the seizure oligonucleotide of about 50 picomole (spacer-5 '-GAT TTG TAT TGATTG AGA TTA AAG-3 ') ([SEQ ID NO:4] is labeled as B among Fig. 4) is combined in shape to be applicable on the hole of the band of test tube of thermal cycling machine or 96 orifice plates.Can utilize 5 ' modification group (for example, between 5 ' end of amino and seizure oligonucleotide, to contain (CH ₂) ₆" at interval " amino of sequence (or similar sequence)) realize to catch combining of oligonucleotide and hole.Perhaps, can will be able to be mixed an end of catching oligonucleotide by 5 ' biotin moiety of flat board in conjunction with the Streptavidin bag so that high avidity is non-covalent.Should notice in the embodiment of this ligase enzyme mediation that the sequence of seizure oligonucleotide is identical with embodiment 1 used seizure oligonucleotide.This sequence is connected the non-influenza virus sequence complementary portion complementation of oligonucleotide ChPB2U with mosaic type.

With the signal oligonucleotide be designed to contain with the test section (for example, latex bead) link coupled sequence 5 '-CTTTAA TCT CAA TCA ATA CAA ATC-3 ' [SEQ ID NO:1], it is added each hole of wrapping the microtiter plate of quilt in advance with seizure oligonucleotide (spacer-5 '-GAT TTG TAT TGA TTG AGA TTA AAG-3 ') [SEQ ID NO:4].The add-on of signal oligonucleotide is caught the oligonucleotide amount about equally with fixed.Can come the accurate amount of definite seizure oligonucleotide that will add by rule of thumb by each test of titration and each batch reagent.

The reaction of ligase enzyme mediation comprises:

1.95 ℃ 2 minutes, make the sex change of target plasmid DNA.

2. make first target sequence (Ch-PB2U) of mosaic type oligonucleotide and first subsequence of target nucleic acid sequence hybridize to form first crossbred wherein 5 ' the Nucleotide complementation of 3 ' Nucleotide of this mosaic type oligonucleotide and this first subsequence.

3. adjoin second subsequence hybridization of this first subsequence to form second crossbred in the second target sequence (PB2D) that makes the first interoperability oligonucleotide and the target nucleic acid sequence.

4. in the presence of the 5 ' Nucleotide of this first subsequence, utilize the Taq dna ligase to connect the mosaic type oligonucleotide shape and the first interoperability oligonucleotide, form first duplex, it comprises first and second subsequences of this target nucleic acid sequence and comprises this mosaic type oligonucleotide simultaneously and the product that is connected of the first interoperability oligonucleotide.

5.95 ℃ make the first duplex sex change separately connect product and target nucleic acid in 30 seconds.

6. the first interoperability sequence that makes custom-designed second kind of interoperability oligonucleotide (B2-E2 among Fig. 4) be connected product with mosaic type oligonucleotide and this is partly hybridized to form reaction product.This custom-designed second kind of interoperability oligonucleotide adds in the time of can finishing in the working cycle of ligase enzyme mediation, perhaps can be present in the reaction mixture when this process begins.

7. the reaction product of above formation has separated contained barrier oligonucleotide sequence in the second interoperability oligonucleotide (being labeled as B2-E2 among Fig. 4).Separate the blocking-up sequence and make the signal oligonucleotide to catch oligonucleotide hybridization, thereby produce the signal (by the partial concn of latex beads) relevant with the target sequence content quantitative with fixed.

Produce typical curve by ligase enzyme mediated detection process from the known control plasmid serial dilution of initial concentration, the signal that produces and typical curve are made comparisons and assess the initial concentration of unknown sample.

Embodiment 4

The rolling circle amplification reaction

Present embodiment has been described the detection and the quantitative assay in the influenza virus PB2 constant gene segment C zone that comprises codon 627.In the present embodiment, will contain the plasmid PCR-TOPO2.1 (hero company) of 986bp DNA inset of PB2 constant gene segment C as target.United States Patent (USP) 5,854 has been described the scheme of initial connection and RCA step in 033.

1.97 ℃, several plasmid serial dilutions of 1 nanogram-300 nanogram are heated in different plastic test tubes made it sex change in 4 minutes, and under the condition of contact that has 5 ' phosphorylation open loop probe to exist incubation, 5 ' and 3 ' of described probe is held identical with the target specificity part that is connected oligonucleotide CH-PB2U and PB2D respectively.The open loop interoperability oligonucleotide of 95 Nucleotide and following sequence coupling: 5 '-AIC AIA GIA GIA TGC AGT TCATAA GAC TCG TCA TGT CTC AGC AGC TTC TAA CGG TCA CTA ATA CGACTC ACT ATA GG TTG CAG CIG CIC CAC CIG-3 ' [SEQ ID NO:7].The concentration of T4 dna ligase in the reaction mixture (New England Biolabs, Inc. (US) Massachusetts, United States of America (New England Biolabs)) is every mL damping fluid 5 units, and described damping fluid contains 10mM Tris-HCl (pH7.5), 0.20MNaCl, 10mM MgCl ₂, 2mM ATP.The concentration of open loop probe is 80nM.Total reaction volume is 40 microlitres.37 ℃ connect 25 minutes.

2. get 25 μ L from each test tube, mix, contain 50mMTris-HCl (pH7.5), 10mM MgCl in the described damping fluid with isopyknic damping fluid ₂, 1mM DTT, 400 each dTTP of μ M, dATP, dGTP, dCTP, also containing concentration is 0.2 μ M, has 42 base mosaic type rolling-circle replication primers of following sequence: 5 '-CTTTAA TCT CAA TCA ATA CAA ATC GCT GAG ACA TGA CGA GTC-3 ' [SEQ IDNO:8].Certain zone that should note this sequence be with embodiment 1 in be used for identical the caught sequence of caught sequence of RT-PCR.Different zones and open loop interoperability oligonucleotide [SEQ ID NO:7] complementation.

3. add Φ 29 archaeal dna polymerases (160ng/50 μ L), 30 ℃ of incubation reaction mixtures 30 minutes.

4. in above reaction mixture, add and the another kind of interoperability oligonucleotide of new synthetic chain complementary, i.e. reverse primer.The concentration of this primer is 0.2 μ M, its sequence is 5 '-AAT ACG ACT CAC TAT AGG-3 ' [SEQ ID NO:9].Be reflected at 30 ℃ and continue 30 minutes again, synthetic second chain and initial and cyclisation interoperability oligonucleotide complementary sequence reverse complemental also comprise sequence (being used as the barrier sequence) with regional complementarity shown in the SEQ ID NO:8.

Each reaction tube contain with (above-mentioned) complementary of zone shown in the SEQ ID NO:8 fixing catch oligonucleotide [SEQ ID NO:4] and with the identical signal oligonucleotide of embodiment 1 used signal oligonucleotide.Along with second chain is synthetic in step 4, synthesized the barrier sequence, this sequence has separated zone shown in the SEQ ID NO:8, and the bonded competition that itself and fixed are caught between oligonucleotide sequence [SEQ ID NO:4] and the signal oligonucleotide is less.Along with the accumulation of the second chain product quantity, the quantity of bonded signal oligonucleotide increases.The semaphore that is produced is proportional with the plasmid consumption that adds the initial action person.

6. in another example of RCA, this method is identical with former example, except detecting when reaction finishes in step and the different detections hole that 25 each response sample of μ L is transferred in the micropore flat board.Catch the tested gaging hole of the pre-bag of oligonucleotide with 100 picomole, contain the signal oligonucleotide coupled (100 picomole) in these holes with vitamin H.Use Streptavidin peroxidase (Luo Shi catalog number (Cat.No.) 1089153) and substrate TMB (adopting the method identical) detection institute bonded signal oligonucleotide concentration then with the foregoing description 2.

Embodiment 5

" terminal point " detects II

Present embodiment has shown another embodiment of end point determination step.As described in embodiment 1, detection reagent and signal reagent are added in 96 orifice plates, wherein every hole 0.01 picomole of fixedly having an appointment is caught oligonucleotide.Can catch mosaic type RT-PCR primer and be 5 '-CTT TAA TCT CAA TCA ATA CAA ATC GAI GTIAGI GAI ACI CAI GG-3 ' [SEQ ID NO:10].The signal oligonucleotide is made the FAM mark shown in embodiment 1, the mole number of its amount in detecting the hole and seizure oligonucleotide is (though various different analyte is wanted titration) about equally.The form of PCR primer such as following [0233] listed (PCR-label primer and PB2 (2) reverse primer).

Shown in embodiment 1, carry out PCR reaction, but begin as target and omitted initial RT (reverse transcription) step with 200 pik cDNA.This is reflected at not comprise during the PCR catches oligonucleotide and signal oligonucleotide ground carries out.After PCR reaction finishes, as hypomere [0233] processing reaction product as described in [0241].

Along with the PCR reaction is carried out, mosaic type can be caught PCR primer (PCR-label primer) and be mixed in the double-stranded product, and it is lower with the efficient of catching competitive oligonucleotide binding signal oligonucleotide.The content of PCR product was high more when reaction finished, and the competition of binding signal oligonucleotide is just weak more.

The concrete reaction conditions that detects H7N7 influenza A virus cDNA is as follows:

Dna profiling (H7N7 influenza A virus cDNA)	200pg
Dna profiling (H7N7 influenza A virus cDNA)	200pg	PCR-label primer: 5 '-CTT TAA TCT CAA TCA ATA CAA ATC GAI GTI AGI GAI ACI CAI GG-3 ' [SEQ ID NO:10]	0.5-1 picomole
PB2 (2) reverse primer: 5 '-AGT ATY CTC ATY CCW GAI CC-3 ' [SEQ ID NO:3]	4 picomole		0.5-1 picomole
	4 picomole	dNTP	0.2mM
Archaeal dna polymerase	1 unit	dNTP	0.2mM
Archaeal dna polymerase	1 unit	MgCl ₂	2mM
The PCR damping fluid	1×	MgCl ₂	2mM
The PCR damping fluid	1×	Add water and reach final volume	50μL

Behind the pcr amplification (35 take turns), with one times of PCR product dilution.

Prepare following mixture:

Anti--label: (5 '-vitamin H-GAT TTG TAT TGA TTG AGA TTA AAG-3 ', [SEQ ID NO:4])	0.01 picomole
	0.01 picomole	Label: (5 '-FAM-CTT TAA TCT CAA TCA ATA CAA ATC-3 ', [SEQ ID NO:1])	0.25 picomole
^*Water will resist-and the cumulative volume of label and label mends extremely	50μL		0.25 picomole
	50μL	The PCR product	50μL

100 μ L reaction mixtures are transferred in each hole of Streptavidin flat board 37 ℃ of incubations 40 minutes.Outwell the mixture in the flat board, with PBS washing 4 times.

In each hole of Streptavidin flat board, add with antibody dilution agent (150mM Tris-HCl, 100mMNaCl, 2%V/V FBS (foetal calf serum) does 1: 1, anti--FAM antibody that 100 μ L (15 milliunit) peroxidase of 000 dilution is coupled, 37 ℃ of incubations 40 minutes.

Outwell the mixture in the flat board, with PBS washing 4 times.

Add 100 μ L BM indigo plant (Luo Shi) substrates in each hole of Streptavidin flat board, incubation is 5 minutes under the room temperature.

With 100 μ L H ₂SO ₄Add each hole of Streptavidin flat board, incubation is 10 minutes under the room temperature.

Detect the absorbancy of 450nm.

As shown in Figure 7, the result of this analysis shows employing end point determination step, and this test can detect H7N7 influenza A virus cDNA delicately, and is good with respect to the signal to noise ratio of background.

Fig. 8 has shown the result who measures the titration experiments of PCR-label primer optimum amount in the above-mentioned test.These results show that the optimum amount of PCT-label primer among the 50 μ L is about 0.5 picomole.Agarose gel electrophoresis and ethidium bromide staining are measured to and adopt this method amplification PCR products to have the big or small (see figure 9) of expection.

The content of every part of patent, patent application and publication that this paper quotes is included this paper in as a reference in full.

This paper quotes any reference and should not be construed as and admit that this reference can be used as the application " prior art ".

The purpose of specification sheets is to describe preferred implementation of the present invention, rather than the present invention is limited to arbitrary embodiment or concrete characteristics combination.Therefore, one skilled in the art will appreciate that using for reference this paper content can make various improvement and change and do not depart from the scope of the present invention in the embodiment of institute's example.All these improvement and change should belong in the scope of the claims of enclosing.

Claims

1. the method for target nucleic acid sequence in the analytical sample, this method comprises:

-in a reaction vessel, merge:

(1) not with the seizure oligonucleotide of this target nucleic acid sequence hybridization (for example, fixed or be free in the solution);

(3) at least a mosaic type oligonucleotide, it comprises:

(i) catch oligonucleotide; Or

(ii) signal oligonucleotide

(b) subsequence of the complementary strand of target nucleic acid sequence, or

(c) at least a mosaic type oligonucleotide; With

(5) comprise the sample of nucleic acid;

-make the inclusion in this reaction vessel carry out the nucleic acid processing reaction, if have target nucleic acid sequence in the sample then form reaction product, wherein the reaction product that so forms comprises first chain and second chain, first chain comprises at least a mosaic type oligonucleotide or its extension products, second chain comprises at least a interoperability oligonucleotide or its extension products, thereby the caught sequence of having blocked the mosaic type oligonucleotide with catch oligonucleotide hybridization, and then make the signal oligonucleotide with catch oligonucleotide hybridization; With

2. the method for claim 1 is characterized in that, described nucleic acid processing reaction is a polymerization dependency nucleic acid processing reaction.

3. the method for claim 1 is characterized in that, it comprises:

B) in the presence of polymerization agent and nucleotide precursor, use the non-hybridization portion of this target nucleic acid sequence to extend the mosaic type oligonucleotide of this first crossbred, thereby formed first duplex that comprises first extension products and target nucleic acid sequence as template;

4. method as claimed in claim 3 is characterized in that, step a)-e) repeats one or many.

5. method as claimed in claim 3 is characterized in that, described polymerization agent is the primer dependent dna-polymerases.

6. method as claimed in claim 3 is characterized in that, the polymerization agent in the step b) is a primer dependency reversed transcriptive enzyme.

7. the method for claim 1 is characterized in that, described polymerization dependency nucleic acid processing reaction comprises:

Ii) make the second target sequence of this first interoperability oligonucleotide and second subsequence hybridization of this target nucleic acid sequence form second crossbred, wherein this second subsequence is adjoined this first subsequence;

Viii) in the presence of polymerization agent and nucleotide precursor, use this first extension products to extend the second interoperability oligonucleotide of the 4th crossbred as template, thus form comprise first extension products and with the reaction product of this first extension products complementary, second extension products.

8. the method for claim 1 is characterized in that, described nucleic acid processing reaction is a ligase enzyme dependency nucleic acid processing reaction.

9. method as claimed in claim 3 is characterized in that it comprises:

The 5 ' Nucleotide of this first subsequence be connected reagent in the presence of, connect this mosaic type oligonucleotide and this first interoperability oligonucleotide, form first duplex, described first duplex comprises first and second subsequences of this target nucleic acid sequence and comprises this mosaic type oligonucleotide simultaneously and the product that is connected of this first interoperability oligonucleotide;

10. method as claimed in claim 3 is characterized in that, comprising:

11. method as claimed in claim 3 is characterized in that, it comprises:

I. make the first target sequence of the first mosaic type oligonucleotide and first subsequence hybridization of target nucleic acid sequence form first crossbred, wherein this first mosaic type oligonucleotide comprise can with the caught sequence of catching oligonucleotide hybridization, and 5 ' the Nucleotide complementation of 3 ' Nucleotide of this first mosaic type oligonucleotide and this first subsequence;

Ii. make second subsequence hybridization of adjoining this first subsequence in the second target sequence of the second mosaic type oligonucleotide and the target nucleic acid sequence form second crossbred, wherein this first mosaic type oligonucleotide comprise can with the caught sequence of signal oligonucleotide hybridization;

Iv. make this first duplex sex change with from target nucleic acid release reaction product.

12., it is characterized in that step 1-5 or a-e or i-iv repeat one or many as each described method among the claim 9-11.

13. a test kit, it is equipped with:

(3) at least a mosaic type oligonucleotide, it comprises:

(i) catch oligonucleotide; Or

(ii) signal oligonucleotide

(b) subsequence of the complementary strand of target nucleic acid sequence, or

(c) at least a mosaic type oligonucleotide.

14. test kit as claimed in claim 13 is characterized in that, it also is equipped with (5) one or more polymerization agents and/or connects reagent.

15., it is characterized in that any one or more in component (1)-(5) adopts lyophilized form as claim 13 or 14 described test kits.

16., it is characterized in that any two in component (1)-(5) or multiple employing form of mixtures as claim 13 or 14 described test kits.

17., it is characterized in that any two in component (1)-(5) or multiple being contained in the different vessels as claim 13 or 14 described test kits.

18. test kit as claimed in claim 13 is characterized in that, described seizure oligonucleotide is fixed on the solid surface.

19. test kit as claimed in claim 13 is characterized in that, described seizure oligonucleotide is fixed on the surface of particulate or pearl.

20. test kit as claimed in claim 13 is characterized in that, described seizure oligonucleotide is fixed on the surface of nano wire.

21. test kit as claimed in claim 13 is characterized in that, described seizure oligonucleotide is fixed on the surface of reaction vessel.

22. test kit as claimed in claim 13 is characterized in that, a plurality of seizure oligonucleotide are fixed to catch the oligonucleotide arrays form.

23. test kit as claimed in claim 22 is characterized in that, described array is the solid phase array.

24. test kit as claimed in claim 22 is characterized in that, described array is a liquid phase array.

25. test kit as claimed in claim 13 is characterized in that, in component (1)-(5) any one or more is provided in reaction vessel.

26. test kit as claimed in claim 25 is characterized in that, described component exists to optimize in advance to be used for the form of each the described method among the claim 1-12 of implementing.

27. test kit as claimed in claim 26 is characterized in that, the final user adds at least a in nucleic acid samples, nucleic acid treat enzyme, mosaic type oligonucleotide and the interoperability oligonucleotide and implements described method in reaction vessel.